Peptide ligands for binding to EphA2

ABSTRACT

A peptide ligand specific for EphA2 comprising a polypeptide comprising three residues selected from cysteine, L-2,3-diaminopropionic acid (Dap), N-beta-alkyl-L-2,3-diaminopropionic acid (N-AlkDap) and N-beta-haloalkyl-L-2,3-diaminopropionic acid (N-HAlkDap), with the proviso that at least one of said three residues is selected from Dap, N-AlkDap or N-HAlkDap, the said three residues being separated by at least two loop sequences, and a molecular scaffold, the peptide being linked to the scaffold by covalent alkylamino linkages with the Dap or N-AlkDap or N-HAlkDap residues of the polypeptide and by thioether linkages with the cysteine residues of the polypeptide when the said three residues include cysteine, such that two polypeptide loops are formed on the molecular scaffold.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application under 35 U.S.C. §371 of International Application No. PCT/EP2019/065993, filed Jun. 18,2019, which claims priority under 35 U.S.C. § 119 to United KingdomApplication No. GB1810316.8, filed Jun. 22, 2018, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 15, 2022, isnamed 178093 SL.txt and is 101,524 bytes in size.

TECHNICAL FIELD

The present invention relates to peptide ligands showing high bindingaffinity to the Eph receptor tyrosine kinase A2 (EphA2). The inventionalso includes drug conjugates comprising said peptides, conjugated toone or more effector and/or functional groups, to pharmaceuticalcompositions comprising said peptide ligands and drug conjugates and tothe use of said peptide ligands and drug conjugates in preventing,suppressing or treating a disease or disorder characterised byoverexpression of EphA2 in diseased tissue (such as a tumour).

In particular, the invention relates to peptide ligands of this typehaving novel chemistries for forming two or more bonds between a peptideand a scaffold molecule.

BACKGROUND OF THE INVENTION

Different research teams have previously tethered peptides to scaffoldmoieties by forming two or more thioether bonds between cysteineresidues of the peptide and suitable functional groups of a scaffoldmolecule. For example, methods for the generation of candidate drugcompounds by linking cysteine-containing peptides to a molecularscaffold as for example tris(bromomethyl) benzene are disclosed in WO2004/077062 and WO 2006/078161.

The advantage of utilising cysteine thiols for generating covalentthioether linkages in order to achieve cyclisation resides is theirselective and biorthogonal reactivity. Thiol-containing linear peptidesmay be cyclised with a thiol-reactive scaffold compound such as 1, 3, 5tris-bromomethylbenzene (TBMB) to form Bicyclic Peptides, and theresultant product contains three thioethers at the benzylic locations.The overall reaction of the linear peptide with TBMB to form a loopedbicyclic peptide with thioether linkages is shown in FIG. 1 .

A need exists for alternative chemistries for coupling peptides toscaffold moieties to form looped peptide structures employing suitablereplacements of the thioether moiety, thereby achieving compatibilitywith different peptides, changes in physiochemical properties such asimproved solubility, changes in biodistribution and other advantages.

WO2011/018227 describes a method for altering the conformation of afirst peptide ligand or group of peptide ligands, each peptide ligandcomprising at least two reactive groups separated by a loop sequencecovalently linked to a molecular scaffold which forms covalent bondswith said reactive groups, to produce a second peptide ligand or groupof peptide ligands, comprising assembling said second derivative orgroup of derivatives from the peptide(s) and scaffold of said firstderivative or group of derivatives, incorporating one of: (a) alteringat least one reactive group; or (b) altering the nature of the molecularscaffold; or (c) altering the bond between at least one reactive groupand the molecular scaffold; or any combination of (a), (b) or (c).

Our earlier pending applications PCT/EP2017/083953 and PCT/EP2017/083954filed 20 Dec. 2017 describe bicycle peptides in which one or morethioether linkages to the scaffold molecule have been replaced byalkylamino linkages.

Eph receptor tyrosine kinases (Ephs) belong to a large group of receptortyrosine kinases (RTKs), kinases that phosphorylate proteins on tyrosineresidues. Ephs and their membrane bound ephrin ligands (ephrins) controlcell positioning and tissue organization (Poliakov et al. (2004) DevCell 7, 465-80). Functional and biochemical Eph responses occur athigher ligand oligomerization states (Stein et al. (1998) Genes Dev 12,667-678).

Among other patterning functions, various Ephs and ephrins have beenshown to play a role in vascular development. Knockout of EphB4 andephrin-B2 results in a lack of the ability to remodel capillary bedsinto blood vessels (Poliakov et al., supra) and embryonic lethality.Persistent expression of some Eph receptors and ephrins has also beenobserved in newly-formed, adult micro-vessels (Brantley-Sieders et al.(2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

The de-regulated re-emergence of some ephrins and their receptors inadults also has been observed to contribute to tumor invasion,metastasis and neo-angiogenesis (Nakamoto et al. (2002) Microsc Res Tech59, 58-67; Brantley-Sieders et al., supra). Furthermore, some Eph familymembers have been found to be over-expressed on tumor cells from avariety of human tumors (Brantley-Sieders et al., supra); Marme (2002)Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

EPH receptor A2 (ephrin type-A receptor 2) is a protein that in humansis encoded by the EPHA2 gene.

EphA2 is upregulated in multiple cancers in man, often correlating withdisease progression, metastasis and poor prognosis e.g.: breast(Zelinski et al (2001) Cancer Res. 61, 2301-2306; Zhuang et al (2010)Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS One 6,e24426), lung (Brannan et al (2009) Cancer Prev Res (Phila) 2,1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo et al(2013) J Thorac Oncol. 8, 301-308), gastric (Nakamura et al (2005)Cancer Sci. 96, 42-47; Yuan et al (2009) Dig Dis Sci 54, 2410-2417),pancreatic (Mudali et al (2006) Clin Exp Metastasis 23, 357-365),prostate (Walker-Daniels et al (1999) Prostate 41, 275-280), liver (Yanget al (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky et al(2005) Mol Cancer Res. 3, 541-551; Li et al (2010) Tumour Biol. 31,477-488).

The full role of EphA2 in cancer progression is still not definedalthough there is evidence for interaction at numerous stages of cancerprogression including tumour cell growth, survival, invasion andangiogenesis. Downregulation of EphA2 expression suppresses tumourcancer cell propagation (Binda et al (2012) Cancer Cell 22, 765-780),whilst EphA2 blockade inhibits VEGF induced cell migration (Hess et al(2001) Cancer Res. 61, 3250-3255), sprouting and angiogenesis (Cheng etal (2002) Mol Cancer Res. 1, 2-11; Lin et al (2007) Cancer 109, 332-40)and metastatic progression (Brantley-Sieders et al (2005) FASEB J. 19,1884-1886).

An antibody drug conjugate to EphA2 has been shown to significantlydiminish tumour growth in rat and mouse xenograft models (Jackson et al(2008) Cancer Research 68, 9367-9374) and a similar approach has beentried in man although treatment had to be discontinued for treatmentrelated adverse events (Annunziata et al (2013) Invest New drugs 31,77-84).

Our earlier pending applications GB1707734.8 filed on 15 May 2017, andGB1721259.8 and GB1721265.5 both filed 19 Dec. 2017, describe bicyclepeptide ligands having high binding affinity for EphA2. Theseapplications further describe conjugates of the peptide ligands withtherapeutic agents, in particular with cytotoxic agents.

SUMMARY OF THE INVENTION

The present inventors have found that replacement of thioether linkagesin looped peptides having affinity for EphA2 by alkylamino linkagesresults in looped peptide conjugates that display similar affinities toEphA2 as the corresponding conjugates made with all thioether linkages.The replacement of thioether linkages by alkylamino linkages is expectedto result in improved solubility and/or improved oxidation stability ofthe conjugates according to the present invention.

Accordingly, in a first aspect the present invention provides a peptideligand specific for EphA2 comprising a polypeptide comprising threeresidues selected from cysteine, L-2,3-diaminopropionic acid (Dap),N-beta-alkyl-L-2,3-diaminopropionic acid (N-AlkDap) andN-beta-haloalkyl-L-2,3-diaminopropionic acid (N-HAlkDap), with theproviso that at least one of said three residues is selected from Dap,N-AlkDap or N-HAlkDap, the said three residues being separated by atleast two loop sequences, and a molecular scaffold, the peptide beinglinked to the scaffold by covalent alkylamino linkages with the Dap orN-AlkDap or N-HAlkDap residues of the polypeptide and by thioetherlinkages with the cysteine residues of the polypeptide when the saidthree residues include cysteine, such that two polypeptide loops areformed on the molecular scaffold.

Suitably, the peptide ligand comprises an amino acid sequence selectedfrom:A₁-X₁-A₂-X₂-A₃

wherein:

A₁, A₂, and A₃ are independently cysteine, L-2,3-diaminopropionic acid(Dap), N-beta-alkyl-L-2,3-diaminopropionic acid (N-AlkDap), orN-beta-haloalkyl-L-2,3-diaminopropionic acid (N-HAlkDap), provided thatat least one of A₁, A₂, and A₃ is Dap, N-AlkDap or N-HAlkDap; and

X₁ and X₂ represent the amino acid residues between the Cysteine, Dap,N-AlkDap or N-HAlkDap residues, wherein each of X₁ and X₂ independentlyis a loop sequence of 4, 5, 6 or 7 amino acid residues.

It can be seen that the derivatives of the invention comprise a peptideloop coupled to a scaffold by at least one alkylamino linkage to Dap orN-AlkDap of N-HAlkDap residues and up to two thioether linkages tocysteine.

The prefix “alkyl” in N-AlkDap and N-HAlkDap refers to an alkyl grouphaving from one to four carbon atoms, preferably methyl. The prefix“halo” is used in this context in its normal sense to signify alkylgroups having one or more, suitably one, fluoro-, chloro-, bromo- oriodo-substituents.

When cysteine is present, the thioether linkage(s) provides an anchorduring formation of the cyclic peptides as explained further below. Inthese embodiments, the thioether linkage is suitably a central linkageof the bicyclic peptide conjugate, i.e. in the peptide sequence tworesidues forming alkylamino linkages in the peptide are spaced from andlocated on either side of a cysteine residue forming the thioetherlinkage. The looped peptide structure is therefore a Bicycle peptideconjugate having a central thioether linkage and two peripheralalkylamino linkages. In alternative embodiments, the thioether linkageis placed at the N-terminus or C-terminus of the peptides, the centrallinkage and the other terminal linkage being selected from Dap, N-AlkDapor N-HAlkDap.

In embodiments of the invention all three of A₁, A₂, and A₃ may suitablybe Dap or N-AlkDap or N-HAlkDap. In these embodiments, the peptideligands of the invention are suitably Bicycle conjugates having acentral alkylamino linkage and two peripheral alkylamino linkages, thepeptide forming two loops sharing the central alkylamino linkage. Inthese embodiments, A₁, A₂, and A₃ are suitably all selected fromN-AlkDap or N-HAlkDap, most suitably N-AlkDap, because of favourablereaction kinetics with the alkylated Daps.

In embodiments, the peptide ligand of the present invention additionallycomprises one or more modifications selected from: N-terminal and/orC-terminal modifications; replacement of one or more amino acid residueswith one or more non-natural amino acid residues (such as replacement ofone or more polar amino acid residues with one or more isosteric orisoelectronic amino acids; replacement of one or more hydrophobic aminoacid residues with other non-natural isosteric or isoelectronic aminoacids); addition of a spacer group; replacement of one or more oxidationsensitive amino acid residues with one or more oxidation resistant aminoacid residues; replacement of one or more amino acid residues with analanine, replacement of one or more L-amino acid residues with one ormore D-amino acid residues; N-alkylation of one or more amide bondswithin the bicyclic peptide ligand; replacement of one or more peptidebonds with a surrogate bond; peptide backbone length modification;substitution of the hydrogen on the α-carbon of one or more amino acidresidues with another chemical group, and post-synthetic bioorthogonalmodification of amino acids such as cysteine, lysine, glutamate andtyrosine with suitable amine, thiol, carboxylic acid and phenol-reactivereagents.

Suitably, these embodiments may comprise an N-terminal modificationusing suitable amino-reactive chemistry, and/or C-terminal modificationusing suitable carboxy-reactive chemistry. For example, the N-terminalmodification may comprise the addition of a molecular spacer group whichfacilitates the conjugation of effector groups and retention of potencyof the bicyclic peptide to its target. The spacer group is suitably anoligopeptide group containing from about 5 to about 30 amino acids, suchas an Ala, G-Sar10-A (SEQ ID NO: 1) group or bAla-Sar10-A (SEQ ID NO: 2)group. Alternatively or additionally, the N-terminal and/or C-terminalmodification comprises addition of a cytotoxic agent.

In all of the peptide sequences defined herein, one or more tyrosineresidues may be replaced by phenylalanine. This has been found toimprove the yield of the bicycle peptide product during base-catalyzedcoupling of the peptide to the scaffold molecule.

Suitably, the peptide ligand of the invention is a high affinity binderof the human, mouse and dog EphA2 hemopexin domain. Suitably the bindingaffinity k_(i) is less than about 500 nM, less than about 100 nM, lessthan about 50 nM, less than about 25 nM, or less than about 10 nM. Thebinding affinity in the context of this specification is the bindingaffinity as measured by the methods described below.

Suitably, the peptide ligand of the invention is selective for EphA2,but does not cross-react with EphA1, EphA3 or EphA4. Suitably, thebinding affinity ki with each of these ligands is greater than about 500nM, greater than about 1000 nM, or greater than about 10000 nM.

Suitably, the scaffold comprises a (hetero)aromatic or (hetero)alicyclicmoiety. Suitably, the scaffold comprises a tris-substituted(hetero)aromatic or (hetero)alicyclic moiety, for example atris-methylene substituted (hetero)aromatic or (hetero)alicyclic moiety.The (hetero)aromatic or (hetero)alicyclic moiety is suitably asix-membered ring structure, preferably tris-substituted such that thescaffold has a 3-fold symmetry axis. Thus, in certain preferredembodiments, the scaffold is 1,3,5-tris-methylenebenzene scaffold, forexample obtained by reacting the peptide with1,3,5-tris-(bromomethyl)benzene (TBMB). In other preferred embodiments,the scaffold is a 1,3,5-tris-(acetamido)benzene group, which may bederived by coupling the peptide to 1,3,5-tris-(bromoacetamido)benzene(TBAB) as described further below.

The reactive sites are also suitable for forming thioether linkages withthe —SH groups of cysteine in embodiments where the third residue iscysteine. The —SH group of cysteine is highly nucleophilic, and in theseembodiments it is expected to react first with the electrophilic centresof the scaffold molecule to anchor the peptide to the scaffold molecule,whereafter the amino groups react with the remaining electrophiliccentres of the scaffold molecule to form the looped peptide ligand.

In embodiments, the peptide has protecting groups on nucleophilic groupsother than the amino groups and —SH groups (when present) intended forforming the alkylamino linkages.

Suitably, the peptide ligands of the invention may be made by a methodthat comprises reacting, in a nucleophilic substitution reaction, thepeptide as defined herein with a scaffold molecule having three or moreleaving groups.

In alternative methods, the compounds of the present invention could bemade converting two or more side chain groups of the peptide to leavinggroups, followed by reacting the peptide, in a nucleophilic substitutionreaction, with a scaffold molecule having two or more amino groups.

The nucleophilic substitution reactions may be performed in the presenceof a base, for example where the leaving group is a conventional anionicleaving group. The present inventors have found that the yields ofcyclised peptide ligands can be greatly increased by suitable choice ofsolvent and base for the nucleophilic substitution reaction, andfurthermore that the preferred solvent and base are different from theprior art solvent and base combinations that involve only the formationof thioether linkages. In particular, the present inventors have foundthat improved yields are achieved when using a trialkylamine base, i.e.a base of formula NR₁R₂R₃, wherein R₁, R₂ and R₃ are independently C1-C5alkyl groups, suitably C2-C4 alkyl groups, in particular C2-C3 alkylgroups. Especially suitable bases are triethylamine anddiisopropylethylamine (DIPEA). These bases have the property of beingonly weakly nucleophilic, and it is thought that this property accountsfor the fewer side reactions and higher yields observed with thesebases. The present inventors have further found that the preferredsolvents for the nucleophilic substitution reaction are polar and proticsolvents, in particular MeCN/H₂O (50:50).

In a further aspect, the present invention provides a drug conjugatecomprising the peptide ligand according to the invention conjugated toone or more effector and/or functional groups such as a cytotoxic agentor a metal chelator.

Suitably, the conjugate has the cytotoxic agent linked to the peptideligand by a cleavable bond, such as a disulphide bond. Suitably, thecytotoxic agent is selected from DM1 or MMAE.

In embodiments, the drug conjugate has the following structure:

-   -   wherein: R₁, R₂, R₃ and R₄ represent hydrogen or C1-C6 alkyl        groups;    -   Toxin refers to any suitable cytotoxic agent;    -   Bicycle represents the looped peptide structure;    -   n represents an integer selected from 1 to 10; and    -   m represents an integer selected from 0 to 10.

Suitably, either: R₁, R₂, R₃ and R₄ are all H; or R₁, R₂, R₃ are all Hand R₄=methyl; or R₁, R₂=methyl and R₃, R₄=H; or R₁, R₃=methyl and R₂,R₄=H; or R₁, R₂=H and R₃, R₄=C1-C6 alkyl.

The linker between the toxin and the bicycle peptide may comprise atriazole group formed by click-reaction between an azide-functionalizedtoxin and an alkyne-functionalized bicycle peptide structure (orvice-versa). In other embodiments, the bicycle peptide may contain anamide linkage formed by reaction between a carboxylate-functionalizedtoxin and the N-terminal amino group of the bicycle peptide.

The linker between the toxin and the bicycle peptide may comprise acathepsin-cleavable group to provide selective release of the toxinwithin the target cells. A suitable cathepsin-cleavable group isvaline-citrulline.

The linker between the toxin and the bicycle peptide may comprise one ormore spacer groups to provide the desired functionality, e.g. bindingaffinity or cathepsin cleavability, to the conjugate. A suitable spacergroup is para-amino benzyl carbamate (PABC) which may be locatedintermediate the valine-citrulline group and the toxin moiety. PABC is aso-called self-immolating group that spontaneously breaks away from thetoxin after cleavage of the cleavable group.

Thus, in embodiments, the bicycle peptide-drug conjugate may have thefollowing structure made up of Toxin-PABC-cit-val-triazole-Bicycle:

In further embodiments, the bicycle peptide-drug conjugate may have thefollowing structure made up of Toxin-PABC-cit-val-dicarboxylate-Bicycle:

Wherein (alk) is an alkylene group of formula C_(n)H_(2n) wherein n isfrom 1 to 10 and may be linear or branched, suitably (alk) isn-propylene or n-butylene.

In another aspect, the invention further provides a kit comprising atleast a peptide ligand or conjugate according to the present invention.

In a still further aspect, the present invention provides a compositioncomprising a peptide ligand or conjugate of the present invention, and apharmaceutically acceptable carrier, diluent or excipient.

Moreover, the present invention provides a method for the treatment ofdisease using a peptide ligand, conjugate, or a composition according tothe present invention. Suitably, the disease is a neoplastic disease,such as cancer.

In a further aspect, the present invention provides a method for thediagnosis, including diagnosis of disease using a peptide ligand, or acomposition according to the present invention. Thus in general thebinding of an analyte to a peptide ligand may be exploited to displacean agent, which leads to the generation of a signal on displacement. Forexample, binding of analyte (second target) can displace an enzyme(first target) bound to the peptide ligand providing the basis for abinding assay, especially if the enzyme is held to the peptide ligandthrough its active site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reaction scheme for preparation of thioether-linkedbicyclic peptide ligands according to the prior art;

FIG. 2 shows a schematic structure of a reference bicyclic peptideligand exhibiting specific binding to EphA2.

FIG. 3 shows a schematic structure of a first bicyclic peptide ligandaccording to the present invention;

FIG. 4 shows a schematic structure of a second bicyclic peptide ligandaccording to the present invention;

FIG. 5 shows a schematic structure of a third bicyclic peptide ligandaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry.

Standard techniques are used for molecular biology, genetic andbiochemical methods (see Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.; Ausubel et al., Short Protocols in MolecularBiology (1999) 4th ed., John Wiley & Sons, Inc.), which are incorporatedherein by reference.

The present invention provides a looped peptide structure as defined inclaim 1 comprising two peptide loops subtended between three linkages onthe molecular scaffold, the central linkage being common to the twoloops. The central linkage may be a thioether linkage formed to acysteine residue of the peptide, or it is an alkylamino linkage formedto a Dap or N-AlkDap or N-HalkDap residue of the peptide. The two outerlinkages are suitably alkylamino linkages formed to Dap or N-AlkDap orN-HalkDap residues of the peptide, or one of the outer linkages may be athioether linkage formed to a cysteine residue of the peptide.

In one embodiment, the peptide ligands of the invention are fullycross-reactive with murine, dog, cynomolgus and human EphA2. In a yetfurther embodiment, the peptide ligands of the invention are selectivefor EphA2, but do not cross-react with EphA1, EphA3 or EphA4.

Suitably the binding affinity k_(i) for EphA2 is less than about 500 nM,less than about 100 nM, less than about 50 nM, less than about 25 nM, orless than about 10 nM. Suitably, the binding affinity ki with EphA1,EphA3 and/or EphA4 is greater than about 500 nM, greater than about 1000nM, or greater than about 10000 nM.

The amino acid sequences of specific peptide ligands according to thepresent invention are defined in the accompanying claims.

It will be appreciated that modified derivatives of the peptide ligandsas defined herein are within the scope of the present invention.Examples of such suitable modified derivatives include one or moremodifications selected from: N-terminal and/or C-terminal modifications;replacement of one or more amino acid residues with one or morenon-natural amino acid residues (such as replacement of one or morepolar amino acid residues with one or more isosteric or isoelectronicamino acids; replacement of one or more non-polar amino acid residueswith other non-natural isosteric or isoelectronic amino acids); additionof a spacer group; replacement of one or more oxidation sensitive aminoacid residues with one or more oxidation resistant amino acid residues;replacement of one or more amino acid residues with an alanine,replacement of one or more L-amino acid residues with one or moreD-amino acid residues; N-alkylation of one or more amide bonds withinthe bicyclic peptide ligand; replacement of one or more peptide bondswith a surrogate bond; peptide backbone length modification;substitution of the hydrogen on the alpha-carbon of one or more aminoacid residues with another chemical group, modification of amino acidssuch as cysteine, lysine, glutamate/aspartate and tyrosine with suitableamine, thiol, carboxylic acid and phenol-reactive reagents so as tofunctionalise said amino acids, and introduction or replacement of aminoacids that introduce orthogonal reactivities that are suitable forfunctionalisation, for example azide or alkyn-group bearing amino acidsthat allow functionalisation with alkyn or azide-bearing moieties,respectively.

In one embodiment, the modified derivative comprises an N-terminaland/or C-terminal modification. In a further embodiment, wherein themodified derivative comprises an N-terminal modification using suitableamino-reactive chemistry, and/or C-terminal modification using suitablecarboxy-reactive chemistry. In a further embodiment, said N-terminal orC-terminal modification comprises addition of an effector group,including but not limited to a cytotoxic agent, a radiochelator or achromophore.

In an embodiment, the N-terminal modification comprises the addition ofa molecular spacer group which facilitates the conjugation of effectorgroups and retention of potency of the bicyclic peptide to its target.The spacer group is suitably an oligopeptide group containing from about5 to about 30 amino acids, such as an Ala, G-Sar10-A (SEQ ID NO: 1) orbAla-Sar10-A (SEQ ID NO: 2) group. In one embodiment, the spacer groupis selected from bAla-Sar10-A (SEQ ID NO: 2).

In one embodiment, the modified derivative comprises replacement of oneor more amino acid residues with one or more non-natural amino acidresidues. In this embodiment, non-natural amino acids may be selectedhaving isosteric/isoelectronic side chains which are neither recognisedby degradative proteases nor have any adverse effect upon targetpotency.

Alternatively, non-natural amino acids may be used having constrainedamino acid side chains, such that proteolytic hydrolysis of the nearbypeptide bond is conformationally and sterically impeded. In particular,these concern proline analogues, bulky sidechains, C□-disubstitutedderivatives (for example, aminoisobutyric acid, Aib), and cyclo aminoacids, a simple derivative being amino-cyclopropylcarboxylic acid.

In a further embodiment, the non-natural amino acid residuesare selectedfrom: 1-naphthylalanine; 2-naphthylalanine; cyclohexylglycine,phenylglycine; tert-butylglycine; 3,4-dichlorophenylalanine;cyclohexylalanine; and homophenylalanine.

In a yet further embodiment, the non-natural amino acid residues areselected from: 1-naphthylalanine; 2-naphthylalanine; and3,4-dichlorophenylalanine. These substitutions enhance the affinitycompared to the unmodified wildtype sequence.

In a yet further embodiment, the non-natural amino acid residues areselected from: 1-naphthylalanine. This substitution provided thegreatest level of enhancement of affinity (greater than 7 fold) comparedto wildtype.

In one embodiment, the modified derivative comprises replacement of oneor more oxidation sensitive amino acid residues with one or moreoxidation resistant amino acid residues. In a further embodiment, themodified derivative comprises replacement of a tryptophan residue with anaphthylalanine or alanine residue. This embodiment provides theadvantage of improving the pharmaceutical stability profile of theresultant bicyclic peptide ligand.

In one embodiment, the modified derivative comprises replacement of oneor more charged amino acid residues with one or more hydrophobic aminoacid residues. In an alternative embodiment, the modified derivativecomprises replacement of one or more hydrophobic amino acid residueswith one or more charged amino acid residues. The correct balance ofcharged versus hydrophobic amino acid residues is an importantcharacteristic of the bicyclic peptide ligands. For example, hydrophobicamino acid residues influence the degree of plasma protein binding andthus the concentration of the free available fraction in plasma, whilecharged amino acid residues (in particular arginine) may influence theinteraction of the peptide with the phospholipid membranes on cellsurfaces. The two in combination may influence half-life, volume ofdistribution and exposure of the peptide drug, and can be tailoredaccording to the clinical endpoint. In addition, the correct combinationand number of charged versus hydrophobic amino acid residues may reduceirritation at the injection site (if the peptide drug has beenadministered subcutaneously).

In one embodiment, the modified derivative comprises replacement of oneor more L-amino acid residues with one or more D-amino acid residues.This embodiment is believed to increase proteolytic stability by sterichindrance and by a propensity of D-amino acids to stabilise □-turnconformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

In all of the peptide sequences defined herein, one or more tyrosineresidues may be replaced by phenylalanine. This has been found toimprove the yield of the bicycle peptide product during base-catalyzedcoupling of the peptide to the scaffold molecule.

In one embodiment, the modified derivative comprises removal of anyamino acid residues and substitution with alanines. This embodimentprovides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serveto deliberately improve the potency or stability of the peptide. Furtherpotency improvements based on modifications may be achieved through thefollowing mechanisms:

-   -   Incorporating hydrophobic moieties that exploit the hydrophobic        effect and lead to lower off rates, such that higher affinities        are achieved;    -   Incorporating charged groups that exploit long-range ionic        interactions, leading to faster on rates and to higher        affinities (see for example Schreiber et al, Rapid,        electrostatically assisted association of proteins (1996),        Nature Struct. Biol. 3, 427-31); and    -   Incorporating additional constraint into the peptide, by for        example constraining side chains of amino acids correctly such        that loss in entropy is minimal upon target binding,        constraining the torsional angles of the backbone such that loss        in entropy is minimal upon target binding and introducing        additional cyclisations in the molecule for identical reasons.

(for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010),16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16,4399-418).

The present invention includes all pharmaceutically acceptable(radio)isotope-labeled compounds of the invention, i.e. compounds offormula (II), wherein one or more atoms are replaced by atoms having thesame atomic number, but an atomic mass or mass number different from theatomic mass or mass number usually found in nature, and compounds offormula (I1), wherein metal chelating groups are attached (termed“effector”) that are capable of holding relevant (radio)isotopes, andcompounds of formula (1), wherein certain functional groups arecovalently replaced with relevant (radio)isotopes or isotopicallylabelled functional groups.

Examples of isotopes suitable for inclusion in the compounds of theinvention comprise isotopes of hydrogen, such as ²H (D) and ³H (T),carbon, such as IT, ¹³C and ¹⁴C, chlorine, such as ³⁶Cl, fluorine, suchas ¹⁸F, iodine, such as ¹²³I, ¹²⁵I and ¹³¹I, nitrogen, such as ¹³N and¹⁵N, oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, sulfur,such as ³⁵S, copper, such as ⁶⁴ Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga,yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as²¹³Bi.

Certain isotopically-labelled compounds of formula (II), for example,those incorporating a radioactive isotope, are useful in drug and/orsubstrate tissue distribution studies, and to clinically assess thepresence and/or absence of the EphA2 target on diseased tissues such astumours and elsewhere. The compounds of formula (II) can further havevaluable diagnostic properties in that they can be used for detecting oridentifying the formation of a complex between a labelled compound andother molecules, peptides, proteins, enzymes or receptors. The detectingor identifying methods can use compounds that are labelled withlabelling agents such as radioisotopes, enzymes, fluorescent substances,luminous substances (for example, luminol, luminol derivatives,luciferin, aequorin and luciferase), etc. The radioactive isotopestritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly usefulfor this purpose in view of their ease of incorporation and ready meansof detection.

Substitution with heavier isotopes such as deuterium, i.e. 2H (D), mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining target occupancy.

Incorporation of isotopes into metal chelating effector groups, such as⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, and ¹⁷⁷Lu can be useful for visualizing tumourspecific antigens employing PET or SPECT imaging.

Incorporation of isotopes into metal chelating effector groups, such as,but not limited to ⁹⁰Y, ¹⁷⁷Lu, and ²¹³Bi, can present the option oftargeted radiotherapy, whereby metal-chelator-bearing compounds offormula (II) carry the therapeutic radionuclide towards the targetprotein and site of action.

Isotopically-labeled compounds of formula (II) can generally be preparedby conventional techniques known to those skilled in the art or byprocesses analogous to those described in the accompanying Examplesusing an appropriate isotopically-labeled reagent in place of thenon-labeled reagent previously employed.

Specificity, in the context herein, refers to the ability of a ligand tobind or otherwise interact with its cognate target to the exclusion ofentities which are similar to the target. For example, specificity canrefer to the ability of a ligand to inhibit the interaction of a humanenzyme, but not a homologous enzyme from a different species. Using theapproach described herein, specificity can be modulated, that isincreased or decreased, so as to make the ligands more or less able tointeract with homologues or paralogues of the intended target.Specificity is not intended to be synonymous with activity, affinity oravidity, and the potency of the action of a ligand on its target (suchas, for example, binding affinity or level of inhibition) are notnecessarily related to its specificity.

Binding activity, as used herein, refers to quantitative bindingmeasurements taken from binding assays, for example as described herein.Therefore, binding activity refers to the amount of peptide ligand whichis bound at a given target concentration.

Multispecificity is the ability to bind to two or more targets.Typically, binding peptides are capable of binding to a single target,such as an epitope in the case of an antibody, due to theirconformational properties. However, peptides can be developed which canbind to two or more targets; dual specific antibodies, for example, asknown in the art as referred to above. In the present invention, thepeptide ligands can be capable of binding to two or more targets and aretherefore multispecific. Suitably, they bind to two targets, and aredual specific. The binding may be independent, which would mean that thebinding sites for the targets on the peptide are not structurallyhindered by the binding of one or other of the targets. In this case,both targets can be bound independently. More generally, it is expectedthat the binding of one target will at least partially impede thebinding of the other.

There is a fundamental difference between a dual specific ligand and aligand with specificity which encompasses two related targets. In thefirst case, the ligand is specific for both targets individually, andinteracts with each in a specific manner. For example, a first loop inthe ligand may bind to a first target, and a second loop to a secondtarget. In the second case, the ligand is non-specific because it doesnot differentiate between the two targets, for example by interactingwith an epitope of the targets which is common to both.

In the context of the present invention, it is possible that a ligandwhich has activity in respect of, for example, a target and anorthologue, could be a bispecific ligand. However, in one embodiment theligand is not bispecific, but has a less precise specificity such thatit binds both the target and one or more orthologues. In general, aligand which has not been selected against both a target and itsorthologue is less likely to be bispecific due to the absence ofselective pressure towards bispecificity. The loop length in thebicyclic peptide may be decisive in providing a tailored binding surfacesuch that good target and orthologue cross-reactivity can be obtained,while maintaining high selectivity towards less related homologues.

If the ligands are truly bispecific, in one embodiment at least one ofthe target specificities of the ligands will be common amongst theligands selected, and the level of that specificity can be modulated bythe methods disclosed herein. Second or further specificities need notbe shared, and need not be the subject of the procedures set forthherein.

The peptide ligand compounds of the invention comprise, consistessentially of, or consist of, the peptide covalently bound to amolecular scaffold. The term “scaffold” or “molecular scaffold” hereinrefers to a chemical moiety that is bonded to the peptide at thealkylamino linkages and thioether linkage (when cysteine is present) inthe compounds of the invention. The term “scaffold molecule” or“molecular scaffold molecule” herein refers to a molecule that iscapable of being reacted with a peptide or peptide ligand to form thederivatives of the invention having alkylamino and, in certainembodiments, also thioether bonds. Thus, the scaffold molecule has thesame structure as the scaffold moiety except that respective reactivegroups (such as leaving groups) of the molecule are replaced byalkylamino and thioether bonds to the peptide in the scaffold moiety.

In embodiments, the scaffold is an aromatic molecular scaffold, i.e. ascaffold comprising a (hetero)aryl group. As used herein, “(hetero)aryl”is meant to include aromatic rings, for example, aromatic rings havingfrom 4 to 12 members, such as phenyl rings. These aromatic rings canoptionally contain one or more heteroatoms (e.g., one or more of N, O,S, and P), such as thienyl rings, pyridyl rings, and furanyl rings. Thearomatic rings can be optionally substituted. “(hetero)aryl” is alsomeant to include aromatic rings to which are fused one or more otheraryl rings or non-aryl rings. For example, naphthyl groups, indolegroups, thienothienyl groups, dithienothienyl, and5,6,7,8-tetrahydro-2-naphthyl groups (each of which can be optionallysubstituted) are aryl groups for the purposes of the presentapplication. As indicated above, the aryl rings can be optionallysubstituted. Suitable substituents include alkyl groups (which canoptionally be substituted), other aryl groups (which may themselves besubstituted), heterocyclic rings (saturated or unsaturated), alkoxygroups (which is meant to include aryloxy groups (e.g., phenoxygroups)), hydroxy groups, aldehyde groups, nitro groups, amine groups(e.g., unsubstituted, or mono- or di-substituted with aryl or alkylgroups), carboxylic acid groups, carboxylic acid derivatives (e.g.,carboxylic acid esters, amides, etc.), halogen atoms (e.g., Cl, Br, andI), and the like.

Suitably, the scaffold comprises a tris-substituted (hetero)aromatic or(hetero)alicyclic moiety, for example a tris-methylene substituted(hetero)aromatic or (hetero)alicyclic moiety. The (hetero)aromatic or(hetero)alicyclic moiety is suitably a six-membered ring structure,preferably tris-substituted such that the scaffold has a 3-fold symmetryaxis.

In embodiments, the scaffold is a tris-methylene (hetero)aryl moiety,for example a 1,3,5-tris methylene benzene moiety. In these embodiments,the corresponding scaffold molecule suitably has a leaving group on themethylene carbons. The methylene group then forms the R₁ moiety of thealkylamino linkage as defined herein. In these methylene-substituted(hetero)aromatic compounds, the electrons of the aromatic ring canstabilize the transition state during nucleophilic substitution. Thus,for example, benzyl halides are 100-1000 times more reactive towardsnucleophilic substitution than alkyl halides that are not connected to a(hetero)aromatic group.

In these embodiments, the scaffold and scaffold molecule have thegeneral formula:

Where LG represents a leaving group as described further below for thescaffold molecule, or LG (including the adjacent methylene group formingthe R₁ moiety of the alkylamino group) represents the alkylamino linkageto the peptide in the conjugates of the invention.

In embodiments, the group LG above may be a halogen such as, but notlimited to, a bromine atom, in which case the scaffold molecule is1,3,5-Tris(bromomethyl)benzene (TBMB). Another suitable molecularscaffold molecule is 2,4,6-tris(bromomethyl) mesitylene. It is similarto 1,3,5-tris(bromomethyl) benzene but contains additionally threemethyl groups attached to the benzene ring. In the case of thisscaffold, the additional methyl groups may form further contacts withthe peptide and hence add additional structural constraint. Thus, adifferent diversity range is achieved than with1,3,5-Tris(bromomethyl)benzene.

Another preferred molecule for forming the scaffold for reaction withthe peptide by nucleophilic substitution is1,3,5-tris(bromoacetamido)benzene (TBAB):

In other embodiments, the scaffold is a non-aromatic molecular scaffold,e.g. a scaffold comprising a (hetero)alicyclic group. As used herein,“(hetero)alicyclic” refers to a homocyclic or heterocyclic saturatedring. The ring can be unsubstituted, or it can be substituted with oneor more substituents. The substituents can be saturated or unsaturated,aromatic or nonaromatic, and examples of suitable substituents includethose recited above in the discussion relating to substituents on alkyland aryl groups. Furthermore, two or more ring substituents can combineto form another ring, so that “ring”, as used herein, is meant toinclude fused ring systems. In these embodiments, the alicyclic scaffoldis preferably 1,1′,1″-(1,3,5-tri azinane-1,3,5-triyOtriprop-2-en-1-one(TATA).

In other embodiments the molecular scaffold may have a tetrahedralgeometry such that reaction of four functional groups of the encodedpeptide with the molecular scaffold generates not more than two productisomers. Other geometries are also possible; indeed, an almost infinitenumber of scaffold geometries is possible, leading to greaterpossibilities for peptide ligand diversification.

The peptides used to form the ligands of the invention comprise Dap orN-AlkDap or N-HAlkDap residues for forming alkylamino linkages to thescaffold. The structure of diaminopropionic acid is analogous to andisosteric that of cysteine that has been used to form thioether bonds tothe scaffold in the prior art, with replacement of the terminal —SHgroup of cysteine by —NH₂:

The term “alkylamino” is used herein in its normal chemical sense todenote a linkage consisting of NH or N(R₃) bonded to two carbon atoms,wherein the carbon atoms are independently selected from alkyl,alkylene, or aryl carbon atoms and R₃ is an alkyl group. Suitably, thealkylamino linkages of the invention comprise an NH moiety bonded to twosaturated carbon atoms, most suitably methylene (—CH₂—) carbon atoms.The alkylamino linkages of the invention have general formula:S—R₁—N(R₃)—R₂—P

Wherein:

-   -   S represents the scaffold core, e.g. a (hetero)aromatic or        (hetero)alicyclic ring as explained further below;    -   R₁ is C1 to C3 alkylene groups, suitably methylene or ethylene        groups, and most suitably methylene (CH₂);    -   R₂ is the methylene group of the Dap or N-AlkDap side chain    -   R₃ is H or C1-4 alkyl including branched alkyl and cycloalkyl,        for example methyl, wherein any of the alkyl groups is        optionally halogenated; and    -   P represents the peptide backbone, i.e. the R₂ moiety of the        above linkage is linked to the carbon atom in the peptide        backbone adjacent to a carboxylic carbon of the Dap or N-AlkDap        or N-HAlkDap residue.

Certain bicyclic peptide ligands of the present invention have a numberof advantageous properties which enable them to be considered assuitable drug-like molecules for injection, inhalation, nasal, ocular,oral or topical administration. Such advantageous properties include:

-   -   Species cross-reactivity. This is a typical requirement for        preclinical pharmacodynamics and pharmacokinetic evaluation;    -   Protease stability. Bicyclic peptide ligands should ideally        demonstrate stability to plasma proteases, epithelial        (“membrane-anchored”) proteases, gastric and intestinal        proteases, lung surface proteases, intracellular proteases and        the like. Protease stability should be maintained between        different species such that a bicycle lead candidate can be        developed in animal models as well as administered with        confidence to humans;    -   Desirable solubility profile. This is a function of the        proportion of charged and hydrophilic versus hydrophobic        residues and intra/inter-molecular H-bonding, which is important        for formulation and absorption purposes; and    -   An optimal plasma half-life in the circulation. Depending upon        the clinical indication and treatment regimen, it may be        required to develop a bicyclic peptide for short exposure in an        acute illness management setting, or develop a bicyclic peptide        with enhanced retention in the circulation, and is therefore        optimal for the management of more chronic disease states. Other        factors driving the desirable plasma half-life are requirements        of sustained exposure for maximal therapeutic efficiency versus        the accompanying toxicology due to sustained exposure of the        agent.

It will be appreciated that salt forms are within the scope of thisinvention, and references to peptide ligands of the present inventioninclude the salt forms of said compounds.

The salts of the present invention can be synthesized from the parentcompound that contains a basic or acidic moiety by conventional chemicalmethods such as methods described in Pharmaceutical Salts: Properties,Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth(Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002.Generally, such salts can be prepared by reacting the free acid or baseforms of these compounds with the appropriate base or acid in water orin an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a widevariety of acids, both inorganic and organic. Examples of acid additionsalts include mono- or di-salts formed with an acid selected from thegroup consisting of acetic, 2,2-dichloroacetic, adipic, alginic,ascorbic (e.g. L-ascorbic), L-aspartic, benzene sulfonic, benzoic,4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic,(+)-(1S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic,citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric,gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic),glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric,hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic),isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic,naphthalene-2-sulfonic, naphthalene-1,5-disulfonic,1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic,palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic,salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric,tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic andvaleric acids, as well as acylated amino acids and cation exchangeresins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulfonic,toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic,naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronicand lactobionic acids. One particular salt is the hydrochloride salt.Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may beanionic (e.g., —COOH may be —COO⁻), then a salt may be formed with anorganic or inorganic base, generating a suitable cation. Examples ofsuitable inorganic cations include, but are not limited to, alkali metalions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitableorganic cations include, but are not limited to, ammonium ion (i.e., NH₄⁺) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺).Examples of some suitable substituted ammonium ions are those derivedfrom: methylamine, ethylamine, diethylamine, propylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the compounds of the present invention contain an amine function,these may form quaternary ammonium salts, for example by reaction withan alkylating agent according to methods well known to the skilledperson. Such quaternary ammonium compounds are within the scope of theinvention.

Several conjugated peptides may be incorporated together into the samemolecule according to the present invention. For example two suchpeptide conjugates of the same specificity can be linked together viathe molecular scaffold, increasing the avidity of the derivative for itstargets. Alternatively, in another embodiment a plurality of peptideconjugates are combined to form a multimer. For example, two differentpeptide conjugates are combined to create a multispecific molecule.Alternatively, three or more peptide conjugates, which may be the sameor different, can be combined to form multispecific derivatives. In oneembodiment multivalent complexes may be constructed by linking togetherthe molecular scaffolds, which may be the same or different.

The peptide ligands of the present invention may be made by a methodcomprising: providing a suitable peptide and a scaffold molecule; andforming the thioether (when cysteine is present) and alkylamino linkagesbetween the peptide and the scaffold molecule.

The peptides for preparation of the peptide ligands of the invention canbe made using conventional solid-phase synthesis from amino acidstarting materials, which may include appropriate protecting groups asdescribed herein. These methods for making peptides are well known inthe art.

Suitably, the peptide has protecting groups on nucleophilic groups otherthan the —SH and amine groups intended for forming the alkylaminolinkages. The nucleophilicity of amino acid side chains has been subjectto several studies, and listed in descending order: thiolate incysteines, amines in Lysine, secondary amine in Histidine andTryptophan, guanidino amines in Arginine, hydroxyls in Serine/Threonine,and finally carboxylates in aspartate and glutamate. Accordingly, insome cases it may be necessary to apply protecting groups to the morenucleophilic groups on the peptide to prevent undesired side reactionswith these groups.

In embodiments, the method comprises: synthesising a peptide havingprotecting groups on nucleophilic groups other than the amine groupsintended for forming the alkylamino linkages and second protectinggroups on the amine groups intended for forming alkylamino linkages,wherein the protecting groups on the amine groups intended for formingalkylamino linkages can be removed under conditions different than forthe protecting groups on the other nucleophilic groups, followed bytreating the peptide under conditions selected to deprotect the aminegroups intended for forming alkylamino linkages without deprotecting theother nucleophilic groups. The coupling reaction to the scaffold is thenperformed, followed by removal of the remaining protecting groups toyield the peptide conjugate.

Suitably, the method comprises reacting, in a nucleophilic substitutionreaction, the peptide having the reactive side chain —SH and aminegroups, with a scaffold molecule having three or more leaving groups.

The term “leaving group” herein is used in its normal chemical sense tomean a moiety capable of nucleophilic displacement by an amine group.Any such leaving group can be used here provided it is readily removedby nucleophilic displacement by amine. Suitable leaving groups areconjugate bases of acids having a pKa of less than about 5. Non-limitingexamples of leaving groups useful in the invention include halo, such asbromo, chloro, iodo, O-tosylate (OTos), O-mesylate (OMes), O-triflate(OTO or O-trimethylsilyl (OTMS).

The nucleophilic substitution reactions may be performed in the presenceof a base, for example where the leaving group is a conventional anionicleaving group. The present inventors have found that the yields ofcyclised peptide ligands can be greatly increased by suitable choice ofsolvent and base (and pH) for the nucleophilic substitution reaction,and furthermore that the preferred solvent and base are different fromthe prior art solvent and base combinations that involve only theformation of thioether linkages. In particular, the present inventorshave found that improved yields are achieved when using a trialkylaminebase, i.e. a base of formula NR₁R₂R₃, wherein R₁, R₂ and R₃ areindependently C1-C5 alkyl groups, suitably C2-C4 alkyl groups, inparticular C2-C3 alkyl groups. Especially suitable bases aretriethylamine and diisopropylethylamine (DIPEA). These bases have theproperty of being only weakly nucleophilic, and it is thought that thisproperty accounts for the fewer side reactions and higher yieldsobserved with these bases. The present inventors have further found thatthe preferred solvents for the nucleophilic substitution reaction arepolar and protic solvents, in particular MeCN/H₂O containing MeCN andH₂O in volumetric ratios from 1:10 to 10:1, suitably from 2:10 to 10:2and more suitably from 3:10 to 10:3, in particular from 4:10 to 10:4.

Additional binding or functional activities may be attached to the N orC terminus of the peptide covalently linked to a molecular scaffold. Thefunctional group is, for example, selected from the group consisting of:a group capable of binding to a molecule which extends the half-life ofthe peptide ligand in vivo, and a molecule which extends the half-lifeof the peptide ligand in vivo. Such a molecule can be, for instance, HSAor a cell matrix protein, and the group capable of binding to a moleculewhich extends the half-life of the peptide ligand in vivo is an antibodyor antibody fragment specific for HSA or a cell matrix protein. Such amolecule may also be a conjugate with high molecular weight PEGs.

In one embodiment, the functional group is a binding molecule, selectedfrom the group consisting of a second peptide ligand comprising apeptide covalently linked to a molecular scaffold, and an antibody orantibody fragment. 2, 3, 4, 5 or more peptide ligands may be joinedtogether. The specificities of any two or more of these derivatives maybe the same or different; if they are the same, a multivalent bindingstructure will be formed, which has increased avidity for the targetcompared to univalent binding molecules. The molecular scaffolds,moreover, may be the same or different, and may subtend the same ordifferent numbers of loops.

The functional group can moreover be an effector group, for example anantibody Fc region.

Attachments to the N or C terminus may be made prior to binding of thepeptide to a molecular scaffold, or afterwards. Thus, the peptide may beproduced (synthetically, or by biologically derived expression systems)with an N or C terminal peptide group already in place. Preferably,however, the addition to the N or C terminus takes place after thepeptide has been combined with the molecular backbone to form aconjugate. For example, Fluorenylmethyloxycarbonyl chloride can be usedto introduce the Fmoc protective group at the N-terminus of the peptide.Fmoc binds to serum albumins including HSA with high affinity, andFmoc-Trp or Fmoc-Lys bind with an increased affinity. The peptide can besynthesised with the Fmoc protecting group left on, and then coupledwith the scaffold through the alkylaminos. An alternative is thepalmitoyl moiety which also binds HSA and has, for example been used inLiraglutide to extend the half-life of this GLP-1 analogue.

Alternatively, a conjugate of the peptide with the scaffold can be made,and then modified at the N-terminus, for example with the amine- andsulfhydryl-reactive linker N-e-maleimidocaproyloxy) succinimide ester(EMCS). Via this linker the peptide conjugate can be linked to otherpeptides, for example an antibody Fc fragment.

The binding function may be another peptide bound to a molecularscaffold, creating a multimer; another binding protein, including anantibody or antibody fragment; or any other desired entity, includingserum albumin or an effector group, such as an antibody Fc region.

Additional binding or functional activities can moreover be bounddirectly to the molecular scaffold.

In embodiments, the scaffold may further comprise a reactive group towhich the additional activities can be bound. Preferably, this group isorthogonal with respect to the other reactive groups on the molecularscaffold, to avoid interaction with the peptide. In one embodiment, thereactive group may be protected, and deprotected when necessary toconjugate the additional activities.

Accordingly, in a further aspect of the invention, there is provided adrug conjugate comprising a peptide ligand as defined herein conjugatedto one or more effector and/or functional groups.

Effector and/or functional groups can be attached, for example, to the Nor C termini of the polypeptide, or to the molecular scaffold.

Appropriate effector groups include antibodies and parts or fragmentsthereof. For instance, an effector group can include an antibody lightchain constant region (CL), an antibody CH1 heavy chain domain, anantibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, orany combination thereof, in addition to the one or more constant regiondomains. An effector group may also comprise a hinge region of anantibody (such a region normally being found between the CH1 and CH2domains of an IgG molecule).

In a further embodiment of this aspect of the invention, an effectorgroup according to the present invention is an Fc region of an IgGmolecule. Advantageously, a peptide ligand-effector group according tothe present invention comprises or consists of a peptide ligand Fcfusion having a tβ half-life of a day or more, two days or more, 3 daysor more, 4 days or more, 5 days or more, 6 days or more or 7 days ormore. Most advantageously, the peptide ligand according to the presentinvention comprises or consists of a peptide ligand Fc fusion having atβ half-life of a day or more.

Functional groups include, in general, binding groups, drugs, reactivegroups for the attachment of other entities, functional groups which aiduptake of the macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate into cells will allow peptidesagainst intracellular targets to be effective. Targets that can beaccessed by peptides with the ability to penetrate into cells includetranscription factors, intracellular signalling molecules such astyrosine kinases and molecules involved in the apoptotic pathway.Functional groups which enable the penetration of cells include peptidesor chemical groups which have been added either to the peptide or themolecular scaffold. Peptides such as those derived from such as VP22,HIV-Tat, a homeobox protein of Drosophila (Antennapedia), e.g. asdescribed in Chen and Harrison, Biochemical Society Transactions (2007)Volume 35, part 4, p 821; Gupta et al. in Advanced Drug DiscoveryReviews (2004) Volume 57 9637. Examples of short peptides which havebeen shown to be efficient at translocation through plasma membranesinclude the 16 amino acid penetratin peptide from DrosophilaAntennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p10444), the 18 amino acid ‘model amphipathic peptide’ (Oehlke et al(1998) Biochim Biophys Acts Volume 1414 p 127) and arginine rich regionsof the HIV TAT protein. Non peptidic approaches include the use of smallmolecule mimics or SMOCs that can be easily attached to biomolecules(Okuyama et al (2007) Nature Methods Volume 4 p 153). Other chemicalstrategies to add guanidinium groups to molecules also enhance cellpenetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p 13585).Small molecular weight molecules such as steroids may be added to themolecular scaffold to enhance uptake into cells.

One class of functional groups which may be attached to peptide ligandsincludes antibodies and binding fragments thereof, such as Fab, Fv orsingle domain fragments. In particular, antibodies which bind toproteins capable of increasing the half-life of the peptide ligand invivo may be used.

RGD peptides, which bind to integrins which are present on many cells,may also be incorporated.

In one embodiment, a peptide ligand-effector group according to theinvention has a tβ half-life selected from the group consisting of: 12hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 daysor more, 5 days or more, 6 days or more, 7 days or more, 8 days or more,9 days or more, 10 days or more, 11 days or more, 12 days or more, 13days or more, 14 days or more, 15 days or more or 20 days or more.Advantageously a peptide ligand-effector group or composition accordingto the invention will have a tβ half life in the range 12 to 60 hours.In a further embodiment, it will have a tβ half-life of a day or more.In a further embodiment still, it will be in the range 12 to 26 hours.

In one particular embodiment of the invention, the functional groupconjugated to the looped peptide is selected from a metal chelator,which is suitable for complexing metal radioisotopes of medicinalrelevance. Such effectors, when complexed with said radioisotopes, canpresent useful agents for cancer therapy. Suitable examples includeDOTA, NOTA, EDTA, DTPA, HEHA, SarAr and others (Targeted Radionuclidetherapy, Tod Speer, Wolters/Kluver Lippincott Williams & Wilkins, 2011).

Possible effector groups also include enzymes, for instance such ascarboxypeptidase G2 for use in enzyme/prodrug therapy, where the peptideligand replaces antibodies in ADEPT.

In one particular embodiment of this aspect of the invention, thefunctional group is selected from a drug, such as a cytotoxic agent forcancer therapy. Suitable examples include: alkylating agents such ascisplatin and carboplatin, as well as oxaliplatin, mechlorethamine,cyclophosphamide, chlorambucil, ifosfamide; Anti-metabolites includingpurine analogs azathioprine and mercaptopurine or pyrimidine analogs;plant alkaloids and terpenoids including vinca alkaloids such asVincristine, Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin andits derivatives etoposide and teniposide; Taxanes, including paclitaxel,originally known as Taxol; topoisomerase inhibitors includingcamptothecins: irinotecan and topotecan, and type II inhibitorsincluding amsacrine, etoposide, etoposide phosphate, and teniposide.Further agents can include antitumour antibiotics which include theimmunosuppressant dactinomycin (which is used in kidneytransplantations), doxorubicin, epirubicin, bleomycin and others.

In one further particular embodiment of the invention according to thisaspect, the cytotoxic agent is selected from DM1 or MMAE.

DM1 is a cytotoxic agent which is a thiol-containing derivative ofmaytansine and has the following structure:

Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent andhas the following structure:

In one embodiment, the cytotoxic agent is linked to the bicyclic peptideby a cleavable bond, such as a disulphide bond. In a further embodiment,the groups adjacent to the disulphide bond are modified to control thehindrance of the disulphide bond, and by this the rate of cleavage andconcomitant release of cytotoxic agent.

Published work established the potential for modifying thesusceptibility of the disulphide bond to reduction by introducing sterichindrance on either side of the disulphide bond (Kellogg et al (2011)Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrancereduces the rate of reduction by intracellular glutathione and alsoextracellular (systemic) reducing agents, consequentially reducing theease by which toxin is released, both inside and outside the cell. Thus,selection of the optimum in disulphide stability in the circulation(which minimises undesirable side effects of the toxin) versus efficientrelease in the intracellular milieu (which maximises the therapeuticeffect) can be achieved by careful selection of the degree of hindranceon either side of the disulphide bond.

The hindrance on either side of the disulphide bond is modulated throughintroducing one or more methyl groups on either the targeting entity(here, the bicyclic peptide) or toxin side of the molecular construct.

Thus, in one embodiment, the cytotoxic agent is selected from a compoundof formula:

-   -   wherein n represents an integer selected from 1 to 10; and    -   R₁ and R₂ independently represent hydrogen or methyl groups.

In one embodiment of the compound of the above formula, n represents 1and R₁ and R₂ both represent hydrogen (i.e. the maytansine derivativeDM1).

In an alternative embodiment of the compound of the above formula, nrepresents 2, R₁ represents hydrogen and R₂ represents a methyl group(i.e. the maytansine derivative DM3).

In one embodiment of the compound, n represents 2 and R₁ and R₂ bothrepresent methyl groups (i.e. the maytansine derivative DM4).

It will be appreciated that the cytotoxic agent can form a disulphidebond, and in a conjugate structure with a bicyclic peptide, thedisulphide connectivity between the thiol-toxin and thiol-bicyclepeptide is introduced through several possible synthetic schemes.

In one embodiment, the bicyclic peptide component of the conjugate hasthe following structure:

-   -   wherein m represents an integer selected from 0 to 10,    -   Bicycle represents any suitable looped peptide structure as        described herein; and    -   R₃ and R₄ independently represent hydrogen or methyl.

Compounds of the above formula where R₃ and R₄ are both hydrogen areconsidered unhindered and compounds of the above formula where one orall of R₃ and R₄ represent methyl are considered hindered.

It will be appreciated that the bicyclic peptide of the above formulacan form a disulphide bond, and in a conjugate structure with acytotoxic agent described above, the disulphide connectivity between thethiol-toxin and thiol-bicycle peptide is introduced through severalpossible synthetic schemes.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptideby the following linker:

-   -   wherein R₁, R₂, R₃ and R₄ represent hydrogen or C1-C6 alkyl        groups;    -   Toxin refers to any suitable cytotoxic agent defined herein;    -   Bicycle represents any suitable looped peptide structure as        described herein;    -   n represents an integer selected from 1 to 10; and    -   m represents an integer selected from 0 to 10.

When R₁, R₂, R₃ and R₄ are each hydrogen, the disulphide bond is leasthindered and most susceptible to reduction. When R₁, R₂, R₃ and R₄ areeach alkyl, the disulphide bond is most hindered and least susceptibleto reduction. Partial substitutions of hydrogen and alkyl yield agradual increase in resistance to reduction, and concomitant cleavageand release of toxin. Preferred embodiments include: R₁, R₂, R₃ and R₄all H; R₁, R₂, R₃ all H and R₄=methyl; R₁, R₂=methyl and R₃, R₄═H; R₁,R₃=methyl and R₂, R₄═H; and R₁, R₂═H, R₃, R₄═C1-C6 alkyl.

In one embodiment, the toxin of compound is a maytansine and theconjugate comprises a compound of the following formula:

-   -   wherein R₁, R₂, R₃ and R₄ are as defined above;    -   Bicycle represents any suitable looped peptide structure as        defined herein;    -   n represents an integer selected from 1 to 10; and    -   m represents an integer selected from 0 to 10.

Further details and methods of preparing the above-described conjugatesof bicycle peptide ligands with toxins are described in detail in ourpublished patent applications WO2016/067035 and WO2017/191460. Theentire disclosure of these applications is expressly incorporated hereinby reference.

The linker between the toxin and the bicycle peptide may comprise atriazole group formed by click-reaction between an azide-functionalizedtoxin and an alkyne-functionalized bicycle peptide structure (orvice-versa). In other embodiments, the bicycle peptide may contain anamide linkage formed by reaction between a carboxylate-functionalizedtoxin and the N-terminal amino group of the bicycle peptide.

The linker between the toxin and the bicycle peptide may comprise acathepsin-cleavable group to provide selective release of the toxinwithin the target cells. A suitable cathepsin-cleavable group isvaline-citrulline.

The linker between the toxin and the bicycle peptide may comprise one ormore spacer groups to provide the desired functionality, e.g. bindingaffinity or cathepsin cleavability, to the conjugate. A suitable spacergroup is para-amino benzyl carbamate (PABC) which may be locatedintermediate the valine-citrulline group and the toxin moiety.

Thus, in embodiments, the bicycle peptide-drug conjugate may have thefollowing structure made up of Toxin-PABC-cit-val-triazole-Bicycle:

In further embodiments, the bicycle peptide-drug conjugate may have thefollowing structure made up of Toxin-PABC-cit-val-dicarboxylate-Bicycle:

wherein (alk) is an alkylene group of formula C_(n)H_(2n) wherein n isfrom 1 to 10 and may be linear or branched, suitably (alk) isn-propylene or n-butylene.

A detailed description of methods for the preparation of peptideligand-drug conjugates according to the present invention is given inour earlier applications WO2016/067035 and PCT/EP2017/083954 filed 20Dec. 2017, the entire contents of which are incorporated herein byreference.

Peptide ligands according to the present invention may be employed in invivo therapeutic and prophylactic applications, in vitro and in vivodiagnostic applications, in vitro assay and reagent applications, andthe like.

In general, the use of a peptide ligand can replace that of an antibody.Derivatives selected according to the invention are of usediagnostically in Western analysis and in situ protein detection bystandard immunohistochemical procedures; for use in these applications,the derivatives of a selected repertoire may be labelled in accordancewith techniques known in the art. In addition, such peptide ligands maybe used preparatively in affinity chromatography procedures, whencomplexed to a chromatographic support, such as a resin. All suchtechniques are well known to one of skill in the art. Peptide ligandsaccording to the present invention possess binding capabilities similarto those of antibodies, and may replace antibodies in such assays.

Diagnostic uses include any uses which to which antibodies are normallyput, including test-strip assays, laboratory assays and immunodiagnosticassays.

Therapeutic and prophylactic uses of peptide ligands prepared accordingto the invention involve the administration of derivatives selectedaccording to the invention to a recipient mammal, such as a human.Substantially pure peptide ligands of at least 90 to 95% homogeneity arepreferred for administration to a mammal, and 98 to 99% or morehomogeneity is most preferred for pharmaceutical uses, especially whenthe mammal is a human. Once purified, partially or to homogeneity asdesired, the selected peptides may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes Iand II, Academic Press, NY).

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate carriers. Typically, thesecarriers include aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, any including saline and/or buffered media. Parenteralvehicles include sodium chloride solution, Ringer's dextrose, dextroseand sodium chloride and lactated Ringer's. Suitablephysiologically-acceptable adjuvants, if necessary to keep a peptidecomplex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude antibodies, antibody fragments and various immunotherapeuticdrugs, such as cyclosporine, methotrexate, adriamycin or cisplatinum,and immunotoxins. Pharmaceutical compositions can include “cocktails” ofvarious cytotoxic or other agents in conjunction with the selectedantibodies, receptors or binding proteins thereof of the presentinvention, or even combinations of selected peptides according to thepresent invention having different specificities, such as peptidesselected using different target derivatives, whether or not they arepooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected antibodies, receptors or binding proteinsthereof of the invention can be administered to any patient inaccordance with standard techniques. The administration can be by anyappropriate mode, including parenterally, intravenously,intramuscularly, intraperitoneally, transdermally, via the pulmonaryroute, or also, appropriately, by direct infusion with a catheter. Thedosage and frequency of administration will depend on the age, sex andcondition of the patient, concurrent administration of other drugs,counter-indications and other parameters to be taken into account by theclinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that use levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpeptide ligands or cocktails thereof may also be administered in similaror slightly lower dosages.

A composition containing a peptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the selectedrepertoires of peptides described herein may be used extracorporeally orin vitro selectively to kill, deplete or otherwise effectively remove atarget cell population from a heterogeneous collection of cells. Bloodfrom a mammal may be combined extracorporeally with the selected peptideligands whereby the undesired cells are killed or otherwise removed fromthe blood for return to the mammal in accordance with standardtechniques.

The invention is further described with reference to the followingexamples.

EXAMPLES

Materials and Methods

Precursor Abbreviations Name Precursor Name CAS Supplier 1Nal 1-Fmoc-3-(1-naphthyl- 96402- Fluorochem Naphthylalanine L-alanine 49-22FuAla 2-Furylalanine Fmoc-L-2-furylalanine 159611- Combi 02-6 Blocks2Nal 2- Fmoc-3-(2-naphthyl)- 112883- Alfa Naphthylalanine L-alanine 43-9Aesar 3,3-DPA 3,3- fmoc-3,3- 189937- Alfa Diphenylalaninediphenylalanine 46-0 Aesar 3,4-DCPhe 3,4- Fmoc-3,4-dichloro-L- 17766-Poly Peptide Dichlorophenyl- phenylalanine 59-5 alanine 3Pal3-(3-Pyridyl)- N-Fmoc-3-(3-pyridyl)- 175453- Fluorochem Alanine Lßnine07-3 4,4-BPA 4,4′- Fmoc-L-4, 4′- 199110- Alfa BiphenylalanineBiphenylalanine 64-0 Aesar 4BenzylPro 4-Benzyl- Fmoc-4-Benzyl-PolyPeptide pyrrolidine-2- pyrrolidine-2- carboxylic acid carboxylicacid 4BrPhe 4- Fmoc-4-Bromo-L- 198561- PolyPeptide Bromophenylalaninephenylalanine 04-5 4FlPro 4-Fluoro- Fmoc-4-fluoro- 203866- PolyPeptidepyrrolidine-2- pyrrolidine-2- 19-7 carboxylic acid carboxylic acid4MeoPhe 4- Fmoc-4- 77128- Iris Methoxyphenylalanine Methoxyphenylalanine72-4 Biotech 4Pal 3-(4-Pyridyl)- N-Fmoc-3-(4-pyridyl)- 169555-Fluorochem Alanine L-alanine 95-7 4PhenylPro 4-Phenyl- Fmoc-4-phenyl-269078- Cambridge pyrrolidine-2- pyrrolidine-2- 71-9 carboxylic acidcarboxylic acid Bioscience Ac Acetyl AC3C 1- 1-(Fmoc- 126705- IrisAminocyclopropane- amino)cyclopropane- 22-4 Biotech 1-carboxyliccarboxylic acid acid AC4C 1-Amino-1- 1-(Fmoc-amino)- 885951- Fluorochemcyclobutanecarboxylic cyclobutylcarboxylic 77-9 acid acid AC5C1-Amino-1- 1-(Fmoc- 117322- Iris cyclopentanecarboxylicamino)cyclopentane- 30-2 Biotech acid carboxylic acid AF488AlexaFluor488 AlexaFluor488-NHS Fisher Ester Scientific Aib 2- Fmoc-α-94744- Fluorochem Aminoisobutyric aminoisobutyric acid 50-0 acid Aza-GlyAzaglycine Aze Azetidine Fmoc-L-azetidine-2- 136552- Combi carboxylicacid 06-2 Blocks β-Ala β-Alanine Fmoc-β-alanine 35737- Fluorochem 10-1C5g Cyclopentylglycine Fmoc-L- 220497- Fluorochem cyclopentylglycine61-0 Cba β-Cyclobutylalanine Fmoc-β-cyclobutyl- 478183- IRIS L-alanine62-9 Biotech GmbH Cpa β- Fmoc-β-cyclopropyl- 214750- FluorochemCyclopropylalanine L-alanine 76-2 Cpg Cyclopropylglycine Fmoc-L-1212257- Apollo cycloproprylglycine 18-5 Scientific DOTA1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid F1 5(6)- Sigmacarboxyfluorescein HArg Homo Arginine Fmoc-L- 401915- FluorochemHomoArg(Pbf)-OH 53-5 HPhe HomoPhenylalanine Fmoc-L- 132684- IrisHomophenylalanine 59-4 Biotech HyP Hydroxyproline Fmoc- 122996- SigmaHydroxyproline(tBu)- 47-8 OH NO2Phe 4-Nitrophenylalanine Fmoc-4-nitro-L-95753- PolyPeptide phenylalanine 55-2 Phg PhenylglycineFmoc-L-phenylglycine 102410- Combi 65-1 Blocks Pip Pipecolic acidFmoc-L-Pipecolic acid 86069- Peptech 86-5 Sar Sarcosine, suchFmoc-Sarcosine-OH 77128- Sigma that Sar_(x) 70-2 represents x Sarresidues tBuGly Tert-leucine Fmoc-L-tert-leucine 132684- Fluorochem 60-7Thi 2-Thienylalanine Fmoc-2-Thienylalanine 130309- Novabioc 35-2 hemThiAz 3-(1,2,4-triazol-1- Fmoc-3-(1,2,4-triazol- 1217449- Sigmayl)-Alanine 1-yl)-Ala-OH 37-0 ΨAla Reduced amide on backbone

In addition, the following non-natural amino acid precursors were usedfor the preparation of the DAP and N-MeDAP modified peptides:

Compound CAS Mw Supplier Fmoc-L- 446847- 440.49 Iris Biotech GMBHDap(Boc,Me)-OH 80-9 Fmoc-Dap(Boc)-OH 162558- 426.46 Sigma Aldrich 25-0

Peptide Synthesis

Peptide synthesis was based on Fmoc chemistry, using a Symphony andSymphonyX peptide synthesiser manufactured by Peptide Instruments and aSyro II synthesiser by MultiSynTech. Standard Fmoc-amino acids wereemployed (Sigma, Merck), with appropriate side chain protecting groups:where applicable standard coupling conditions were used in each case,followed by deprotection using standard methodology. Peptides werepurified by HPLC and following isolation they were modified with1,3,5-tris(bromomethyl)benzene (TBMB, Sigma). For this, linear peptidewas diluted with H₂O up to ˜35 mL, ˜500 μL of 100 mM TBMB inacetonitrile was added, and the reaction was initiated with ˜5 mL of 1 MNH₄HCO₃ in H₂O. The reaction was allowed to proceed for ˜30 −60 min atRT, and quenched with 500 ul of the 1M Cysteine hydrochloride (Sigma)once the reaction had completed (judged by MALDI). Followinglyophilisation, the modified peptide was purified in a Gemini C18 column(Phenomenex) using water/acetonitrile with 0.1% trifluoroacetic acid asmobile phase. Pure fractions containing the correct cyclised materialwere pooled, lyophilised and kept at −20° C. for storage.

All amino acids, unless noted otherwise, were used in theL-configurations.

Biological Data

1. Fluorescence Polarisation Measurements

(a) Direct Binding Assay

Peptides with a fluorescent tag (either fluorescein, SIGMA or AlexaFluor488™, Fisher Scientific) were diluted to 2.5 nM in PBS with 0.01%tween 20 or 50 mM HEPES with 100 mM NaCl and 0.01% tween pH 7.4 (bothreferred to as assay buffer). This was combined with a titration ofprotein in the same assay buffer as the peptide to give 1 nM peptide ina total volume of 254 in a black walled and bottomed low bind low volume384 well plates, typically 54 assay buffer, 104 protein (Table 1) then10 μL fluorescent peptide. One in two serial dilutions were used to give12 different concentrations with top concentrations ranging from 500 nMfor known high affinity binders to 10 μM for low affinity binders andselectivity assays. Measurements were conducted on a BMG PHERAstar FSequipped with an “FP 485 520 520” optic module which excites at 485 nmand detects parallel and perpendicular emission at 520 nm. The PHERAstarFS was set at 25° C. with 200 flashes per well and a positioning delayof 0.1 second, with each well measured at 5 to 10 minute intervals for60 minutes. The gain used for analysis was determined for each tracer atthe end of the 60 minutes where there was no protein in the well. Datawas analysed using Systat Sigmaplot version 12.0. mP values were fit toa user defined quadratic equation to generate a Kd value: f=y min+(ymax−y min)/Lig*((x+Lig+Kd)/2−sqrt((((x+Lig+Kd)/2){circumflex over( )}2)−(Lig*x))). “Lig” was a defined value of the concentration oftracer used.

(b) Competition Binding Assay

Peptides without a fluorescent tag were tested in competition with apeptide with a fluorescent tag and a known Kd (Table 2). Peptides werediluted to an appropriate concentration in assay buffer as described inthe direct binding assay with a maximum of 5% DMSO, then seriallydiluted 1 in 2. Five μL of diluted peptide was added to the platefollowed by 104 of human or mouse EphA2 (Table 1) at a fixedconcentration which was dependent on the fluorescent peptide used (Table2), then 104 fluorescent peptide added. Measurements were conducted asfor the direct binding assay, however the gain was determined prior tothe first measurement. Data analysis was in Systat Sigmaplot version12.0 where the mP values were fit to a user defined cubic equation togenerate a Ki value:f=y min+(y max−ymin)/Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*(4(Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}3){circumflex over( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))/((3*Klig)+42*((Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}0.5*COS(ARCCOS((−2*(Klig+Kcomp+Lig+Comp−Prot*c){circumflex over( )}3+9*(Klig+Kcomp+Lig+Comp−Prot*c)*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)−27*(−1*Klig*Kcomp*Prot*c))/(2*(4(Klig+Kcomp+Lig+Comp−Prot*c){circumflexover( )}2−3*(Kcomp*(Lig−Prot*c)+Klig*(Comp−Prot*c)+Klig*Kcomp)){circumflexover ( )}3){circumflex over( )}0.5)))/3))−(Klig+Kcomp+Lig+Comp−Prot*c)))).

“Lig”, “KLig” and “Prot” were all defined values relating to:fluorescent peptide concentration, the Kd of the fluorescent peptide andEphA2 concentration respectively.

TABLE 1 Ephrin receptors and source Receptor Catalogue (domain) SpeciesFormat/tag Supplier number EphA1 (Ecto) Human Fc fusion R&D systems7146-A1 EphA2 (Ecto) Human C-terminal R&D systems 3035-A2 polyHis EphA2(Ecto) Human C-terminal In-house N/A polyHis EphA2 (Ecto) Mouse Fcfusion R&D 639-A2 Systems EphA2 (Ecto) Mouse C-terminal Sino 50586-polyHis Biological M08H EphA2 (ligand Rat C-terminal In-house N/Abinding) polyHis EphA2 (ligand Dog C-terminal In-house N/A binding)polyHis EphA3 (Ecto) Human Fc fusion R&D systems 6444-A3 EphA3 (Ecto)Human N-terminal In-house N/A polyHis EphA3 (Ecto) Rat C-terminal Sino80465-R08H polyHis Biological EphA4 (Ecto) Human Fc fusion R&D systems6827-A4 EphA4 (Ecto) Human C-terminal Sino 11314-H08H polyHis BiologicalEphA4 (Ecto) Rat C-terminal Sino 80123-R08H polyHis Biological EphA6(Ecto) Human Fc fusion R&D systems 5606-A6 EphA7 (Ecto) Human Fc fusionR&D systems 6756-A7 EphB1 (Ecto) Rat Fc fusion R&D systems 1596-B1 EphB4(Ecto) human C-terminal R&D systems 3038-B4 polyHis

TABLE 2 Final concentrations of fluorescent peptide and EphA2 as usedwith Competition Binding Assays Concentration of Concentration ofConcentration of Fluorescent fluorescent peptide Human EphA2 Mouse EphA2peptide (nM) (nM) (nM) Compound 1 1 300 Compound 12 10 75 Compound 66 130 Compound 18 0.8 (human) 2.4 50 1 (mouse)

The peptide ligands described herein were tested in the above mentionedassays.

Reference Example 1

A first reference Bicyclic Peptide chosen for comparison of thioether toalkylamino scaffold linkage was designated 55-03-05-N233. It is abicycle conjugate of a thioether-forming peptide with a trimethylenebenzene scaffold. The structure of this bicycle derivative is shownschematically in FIG. 2 . The linear peptide before conjugation hassequence:

-   -   [B-Ala][Sar]₁₀H[dD]VPCPWGPFWCPVNRPGC

Conjugation to 1,3,5-tris(bromomethyl)benzene (TBMB, Sigma) was carriedout as follows. The linear peptide was diluted with H₂O up to ˜35 mL,˜500 μL of 100 mM TBMB in acetonitrile was added, and the reaction wasinitiated with 5 mL of 1 M NH₄HCO₃ in H₂O. The reaction was allowed toproceed for ˜30 −60 min at RT, and lyophilised once the reaction hadcompleted (judged by MALDI). Following lyophilisation, the modifiedpeptide was purified with a Gemini C18 column (Phenomenex), and changingthe acid to 0.1% trifluoroacetic acid. Pure fractions containing thecorrect TMB-modified material were pooled, lyophilised and kept at −20°C. for storage.

The resulting Bicycle derivative designated 55-03-05-N233 showed highaffinity to EphA2. The measured affinity (Ki) to EphA2 of the derivativewas 4.12 nM.

Example 1

A bicycle peptide designated 55-03-05-N314 was made corresponding to thebicycle region of the peptide ligand of Reference Example 1, withreplacement of the first and second cysteine residues by DAP residuesforming alkylamino linkages to the TBMB scaffold. The structure of thisderivative is shown schematically in FIG. 3 .

The linear peptide used to form this bicycle was as follows:

-   -   [Ac][B-Ala][Sar]₁₀H[dD]VP[Dap]PWGPFW[Dap]PVNRPGC

Cyclisation with TBMB was performed in a mixture of Acetonitrile/waterin the presence of DIPEA as the base for 1-16 hours, as described inmore detail in PCT/EP2017/083953 and PCT/EP2017/083954 filed 20 Dec.2017. Unlike the cyclisation of Reference Example 1, the yield isrelatively low when using the conventional NaHCO₃ as the base.

The measured Ki with EphA2 was 135.5 nM, which demonstrates that thechange to alkylamino linkages in this example resulted in relativelylittle change in binding affinity relative to the thioether linkedderivative of Reference Example 1.

Example 2

A bicycle peptide designated 55-03-05-N316 was made corresponding to thebicycle region of the peptide ligand of Reference Example 1, withreplacement of the second and third cysteine residues by DAP residuesforming alkylamino linkages to the TBMB scaffold. The structure of thisderivative is shown schematically in FIG. 3 .

The linear peptide used to form this bicycle was as follows:

-   -   [Ac][B-Ala][Sar]₁₀H[dD]VPCPWGPFW[Dap]PVNRPG[Dap]

Cyclisation with TBMB was performed as described in Example 1.

The measured Ki with EphA2 was 604 nM, which demonstrates that thechange to alkylamino linkages in this example preserved a relativelyhigh level of binding affinity relative to the thioether-linkedderivative of Reference Example 1.

Example 3

A bicycle peptide designated 55-03-05-N318 was made corresponding to thebicycle region of the peptide ligand of Reference Example 1, withreplacement of the first and third cysteine residues by DAP residuesforming alkylamino linkages to the TBMB scaffold. The structure of thisderivative is shown schematically in FIG. 4 .

The linear peptide used to form this bicycle was as follows:

-   -   [Ac][B-Ala][Sar]₁₀H[dD]VP[Dap]PWGPFWCPVNRPG[Dap]

Cyclisation with TBMB was performed as described in Example 1.

The measured Ki with EphA2 was 31.5 nM, which demonstrates that thechange to alkylamino linkages in this example resulted in only a minimalchange in binding affinity relative to the thioether-linked derivativeof Reference Example 1.

Reference Example 2

A first reference Bicyclic Peptide chosen for comparison of thioether toalkylamino scaffold linkage was designated 55-03-05-N238. It is abicycle conjugate of a thioether-forming peptide with a trimethylenebenzene scaffold. The linear peptide before conjugation has sequence:

-   -   [B-Ala][Sar]₁₀H[dD]VPC[Aib][1Nal]G[Aib]F [1Nal]CP        [tBuGly]N[HArg]P [dD]C

Conjugation to 1,3,5-tris(bromomethyl)benzene (TBMB, Sigma) was carriedout as described in Example 1.

The resulting Bicycle derivative designated 55-03-05-N238 showed highaffinity to EphA2. The measured affinity (Ki) to EphA2 of the derivativewas 19.7 nM.

Example 4

A bicycle peptide designated 55-03-05-N315 was made corresponding to thebicycle region of the peptide ligand of Reference Example 2, withreplacement of the first and second cysteine residues by DAP residuesforming alkylamino linkages to the TBMB scaffold.

The linear peptide used to form this bicycle was as follows:

-   -   [B-Ala][Sar]₁₀H[dD]VP[Dap][Aib][Mal]G[Aib]F[1Nal][Dap]P[tBuGly]N[HArg]P[dD]C

Cyclisation with TBMB was performed as described in Example 1.

The measured Ki with EphA2 was 640 nM, which demonstrates that thechange to alkylamino linkages in this example preserved a significantbinding affinity relative to the thioether-linked derivative ofReference Example 2.

Example 5

A bicycle peptide designated 55-03-05-N317 was made corresponding to thebicycle region of the peptide ligand of Reference Example 2, withreplacement of the second and third cysteine residues by DAP residuesforming alkylamino linkages to the TBMB scaffold.

The linear peptide used to form this bicycle was as follows:

-   -   [B-Ala][Sar]₁₀H[dD]VPC[Aib][Mal]G[Aib]F[1Nal][Dap]P[tBuGly]N[HArg]P[dD][Dap]

Cyclisation with TBMB was performed as described in Example 1.

The measured Ki with EphA2 was 425 nM, which demonstrates that thechange to alkylamino linkages in this example preserved a significantbinding affinity relative to the thioether-linked derivative ofReference Example 2.

Example 6

A bicycle peptide designated 55-03-05-N319 was made corresponding to thebicycle region of the peptide ligand of Reference Example 2, withreplacement of the second and third cysteine residues by DAP residuesforming alkylamino linkages to the TBMB scaffold.

The linear peptide used to form this bicycle was as follows:

-   -   [B-Ala][Sar]₁₀H[dD]VP [Dap][Aib][Mal]G[Aib]F [1Nal]CP        [tBuGly]N[HArg]P [dD][Dap]

Cyclisation with TBMB was performed as described in Example 1.

The measured Ki with EphA2 was 17 nM, which demonstrates that the changeto alkylamino linkages in this example marginally increases the affinityto EphA2 relative to the thioether-linked derivative of ReferenceExample 2.

Reference Examples A1-A308

The following reference peptide ligands having a TBMB scaffold withthree thioether linkages to cysteine residues of the specified peptidesequences were prepared and evaluated for affinity to EphA2 as describedin detail in our earlier application GB201721265.5 filed 19 Dec. 2017.

In view of the results obtained above in Examples 1-6, it is predictedthat derivatives of the reference examples A1-A308 according to thepresent invention, i.e. having alkylamino linkages in place of one ormore of the thioether linkages in the reference examples, will alsodisplay affinity for EphA2. It is further predicted that derivatives ofthe reference examples B1-B98 having scaffolds other than TBMB, inparticular aromatic scaffolds other than TBMB, will also displayaffinity for EphA2. All such derivatives having affinity for EphA2 aretherefore included within the scope of the present invention.

TABLE 3Biological Assay Data for Reference Peptide Ligands (Direct Binding Assay)Bicycle SEQ Compound ID K_(D), nM ± 95% CI Number Sequence NO: ScaffoldHuman EphA2 Mouse EphA 1 ACMNDWLCSLGWTCA-Sar₆-K(Fl) 3 TBMB107.58 ± 40.83 301 n = 1 2 AF488-G-Sar₁₀-ACMNDWLCSLGWTC 4 TBMB 326 n = 13 ACMNDWLCELGWTCA-Sar₆-K(Fl) 5 TBMB 121.48 ± 50.27 4ACTRQGIWCALGFEPCA-Sar₆-K(Fl) 6 TBMB 163.5 ± 22.54 5ACMNDWLCTLGWSCA-Sar₆-K(Fl) 7 TBMB 142.5 ± 83.3 6ACMNDWLCQLGWTCA-Sar₆-K(Fl) 8 TBMB 54.25 ± 4.8 7ACMNDWLCTLGWTCA-Sar₆-K(Fl) 9 TBMB 74.35 ± 15.97 8ACMNDWLCDLGWRCA-Sar₆-K(Fl) 10 TBMB 118.5 ± 22.54 9ACMNDWLCELGWSCA-Sar₆-K(Fl) 11 TBMB 137.5 ± 49.98 10ACRVSPEYCPFGPVWCAGAAA-Sar₆-K(Fl) 12 TBMB 135.13 ± 59.02 11Fl-G-Sar₅-ACPWGPAWCPVHGKTCA 13 TBMB 263 ± 213.64 12Fl-G-Sar₅-ACPWGPAWCPVNRPGCA 14 TBMB 27.78 ± 8.35 13Ac-ACPWGPAWCPVNRPGCAGAAA-K(Fl) 15 TBMB 29 ± 2.55 14AF488-G-Sar₁₀-ACPWGPAWCPVNRPGCA 16 TBMB 38 n = 1 15Fl-G-Sar₅-ACPWGPMWCPVNRPGCA 17 TBMB 12.6 ± 2.55 16Fl-G-Sar₅-ACPWGPNWCPVNRPGCA 18 TBMB 11.5 ± 1.76 17Fl-G-Sar₅-AGEMACPWGPFWCPVNRPGCA 19 TBMB 3.85 ± 0.1 18Fl-G-Sar₅-ADVTCPWGPFWCPVNRPGCA 20 TBMB 0.93 ± 0.23 4.02 ± 2 19Fl-G-Sar₅-ADVRTCPWGPFWCPVNRPGCA 21 TBMB 4.74 ± 0.51 20Fl-G-Sar₅-ANDVTCPWGPGWCPVNRPGCA 22 TBMB 2.35 ± 0.49 21ACVPQGIWCALQFEPCA-Sar₆-K(Fl) 23 TBMB 59.5 ± 12.78 22ACQKQGLWCALGFEPCA-Sar₆-K(Fl) 24 TBMB 289 ± 74.51 23ACLVNDDCFYMGLCA-Sar₆-K(Fl) 25 TBMB 109.38 ± 20.75

TABLE 4 Biological Assay Data for Reference Peptide Ligands (CompetitionBinding Assay) Human EphA2 (K_(i), nM ± 95% CI) Bicycle SEQ FluorescentPeptide Compound ID Compound Compound Compound Compound Number SequenceNO: Scaffold 66 1 12 18 24 ACMNDWLCSLGWTCA 26 TBMB 82.34 ± 12.8 25Ac-CANDWLCSLGWTC 27 TBMB 328 n = 1 26 Ac-CMNDWLCALGWTC 28 TBMB  71.6 ±3.33 27 Ac-CMNDWLCSAGWTC 29 TBMB 356 n = 1 28 ACMNDWLCQLGWKCA 30 TBMB113 n = 1 29 ACMNDWLCELGWTCA 31 TBMB  134.5 ± 32.34 30 ACMNDWLCQLGWTCA32 TBMB 56.05 ± 3.23 31 ACTQNDWLCSLGWTCA 33 TBMB 151.65 ± 161.4 32ACRNIPTMCPFGPVWCA 34 TBMB 83.4 n = 1 33 ACRVSPEYCPFGPVWCA 35 TBMB  78.53± 35.61 34 ACRVSPEYCPFGPVWCAGAAA 36 TBMB  77.4 ± 8.95 35ACRVSPEYCPFGPTWCA 37 TBMB  43.2 ± 13.33 36 ACRVSPEYCPFGPSWCA 38 TBMB 40.5 ± 5.88 37 ACRVSPEYCPFGPEWCA 39 TBMB  61.25 ± 41.85 38ACRVSPEYCPFGPYWCA 40 TBMB  26.53 ± 16.92 39 ACRVSPEYCPFGPLWCA 41 TBMB 32.11 ± 10.28 40 ACRVSPEYCPFGPDWCA 42 TBMB  55.4 ± 9.41 41ACPWGPAWCPVHGKTCA 43 TBMB  263 n = 1 42 ACPWGPAWCPVRDTNCA 44 TBMB 316 n= 1 43 ACPWGPAWCPVNGARCA 45 TBMB 430 n = 1 44 ACPWGPAWCPVNRPGCA 46 TBMB191.22 ± 29.47  164 n = 1 128.45 ± 28.21 45 ACPWGPAWCPVNRPGCAGAAA 47TBMB 117.13 ± 17.96  99.15 ± 48.71 46 ACPWGPMWCPVNRPGCA 48 TBMB  95.75 ±29.89 47 ACPWGPNWCPVNRPGCA 49 TBMB  78.35 ± 12.64 48 ACPWGPAWCPVRNPCA 50TBMB   284 ± 47.04 49 ACPWGPAWCPVSRVCA 51 TBMB   428 ± 99.96 50ACPWGPAWCPVRSCA 52 TBMB    314 ± 248.92 51 ACPWGPAWCPVKPTCA 53 TBMB 318.5 ± 255.78 52 ACPWGPAWCPVNRNGCA 54 TBMB   168 ± 72.52 53AGEMACPWGPFWCPVNRPGCA 55 TBMB    6 ± 5.54 54 AVHIPCPWGPSWCPVNRPCCA 56TBMB  5.17 ± 2.76  5.13 ± 1.52 55 AEGLPCPWGPFWCPVNRPGCA 57 TBMB  6.15 ±3.43  11.3 ± 2.04 56 ADHACPWGPFWCPVNRPGCA 58 TBMB  5.87 ± 5.09 14.43 ±6.28 57 ADVHCPWGPFWCPVNRPGCA 59 TBMB  1.2 n = 1  0.48 ± 0.15 58ADVTCPWGFFWCPVNRPGCA 60 TBMB  2.65 ± 1.08  1.35 ± 0.23 59AHDVPCPWGPFWCPVNRPGCA 61 TBMB  0.54 ± 0.14 60 ADVRTCPWGPFWCPVNRPGCA 62TBMB  2.5 n = 1 12.63 ± 1.29 61 ANDVTCPWGPFWCPVNRPGCA 63 TBMB  7.3 n = 1 2.93 ± 0.07 62 ARDDPCPWGPFWCPVNRPGCA 64 TBMB  27.96 ± 16.74 16.13 ±0.8  63 ACVPQGIWCALQFEPCA 65 TBMB  82.45 ± 27.07  144 n = 1  92.2 ±21.17 64 ACTTGSIWCALQFEPCA 66 TBMB 63.4 n = 1  410 n = 1 65ACVPQGIWCALRYEPCA 67 TBMB 293 n = 1 229 n = 1

TABLE 5Biological Assay Data for Reference Peptide Ligands (Direct Binding Assay)Bicycle K_(D), nM ± 95% CI Number SEQ ID Human Mouse Compound SequenceNO: Scaffold EphA2 EphA2 66 Fl-G-Sar₅-ACPWGPFWCPVNRPGCA 68 TBMB8.45 ± 0.4 22 n = 1 67 AlexaFluor488-G-Sar₅- 69 TBMB 15.03 ± 1.7251.8 ± 6.27 ACPWGPFWCPVNRPGC 68 AlexaFluor488-(β-Ala)-Sar₁₀- 70 TBMB15.37 ± 2.87 23.4 n = 1 ACPWGPFWCPVNRPGC

TABLE 6Biological Assay Data for Reference Peptide Ligands (Competition Binding Assay)Ki, nM ± 95% CI Bicycle Human EphA2 Compound SEQ Fluorescent PeptideNumber Sequence ID NO: Scaffold Compound 18 Compound 66 69ACPWGPFWCPVNRPGCA 71 TBMB 106.75 ± 44.25 70.08 ± 8.01 70Sar₂-ACPWGPFWCPVNRPGC 72 TBMB 51.81 ± 21.75 20.45 ± 12.84 71Ac-Sar₂-ACPWGPFWCPVNRPGC 73 TBMB 11.87 ± 7.51 72(β-Ala)-Sar₁₀-ACPWGPFWCPVNRPGC 74 TBMB 29.1 ± 5.08 20.98 ± 2.18 73Sar₂-AC(HyP)WGPFWCPVNRPGC 75 TBMB 47.6 ± 18.42 247.5 ± 18.62 74Sar₂-AC(Aib)WGPFWCPVNRPGC 76 TBMB 138.9 ± 88.79 75Sar₂-AC(4FlPro)WGPFWCPVNRPGC 77 TBMB 399.67 ± 90.63 76Sar₂-ACP(1Nal)GPFWCPVNRPGC 78 TBMB 3.5 ± 1.96 16.7 ± 9.68 77Sar₂-ACP(2Nal)GPFWCPVNRPGC 79 TBMB 458.33 ± 222.44 78Sar₂-ACPWG(Aze)FWCPVNRPGC 80 TBMB 403.5 ± 12.74 79Sar₂-ACPWG(HyP)FWCPVNRPGC 81 TBMB 131 ± 22.97 80Sar₂-ACPWG(Aib)FWCPVNRPGC 82 TBMB 120.5 ± 81.34 186.73 ± 94.37 81Sar₂-ACPWG(4FlPro)FWCPVNRPGC 83 TBMB 294 ± 99.6 82Sar₂-ACPWG(Pip)FWCPVNRPGC 84 TBMB 497.33 ± 223.62 83Sar₂-ACPWGPAWCPVNRPGC 85 TBMB 199 n = 1 287.5 ± 197.95 84Sar₂-ACPWGP(4Pal)WCPVNRPGC 86 TBMB 33.5 ± 0.98 81.47 ± 68.95 85Sar₂-ACPWGP(4BrPhe)WCPVNRPGC 87 TBMB 174.5 ± 20.58 86Sar₂-ACPWGP(4MeoPhe)WCPVNRPGC 88 TBMB 274.5 ± 36.26 87Sar₂-ACPWGP(HPhe)WCPVNRPGC 89 TBMB 162 n = 1 281.2 ± 154.82 88Sar₂-ACPWGP(4,4-BPA)WCPVNRPGC 90 TBMB 182.67 ± 99.5 89Sar₂-ACPWGP(NO2Phe5)WCPVNRPGC 91 TBMB 289.5 ± 93.1 90Sar₂-ACPWGP(3,4-DCPhe)WCPVNRPGC 92 TBMB 361 ± 25.48 91Sar₂-ACPWGPYWCPVNRPGC 93 TBMB 137.63 ± 104.2 92Sar₂-ACPWGP(3Pal)WCPVNRPGC 94 TBMB 165 ± 27.44 93Sar₂-ACPWGP(Phg)WCPVNRPGC 95 TBMB 411.5 ± 128.38 94Sar₂-ACPWGP(1Nal)WCPVNRPGC 96 TBMB 196.5 ± 6.86 95Sar₂-ACPWGP(2Nal)WCPVNRPGC 97 TBMB 362.5 ± 110.74 96Sar₂-ACPWGPF(1Nal)CPVNRPGC 98 TBMB 31.3 ± 24.11 68.13 ± 35.66 97Sar₂-ACPWGPFWC(Aze)VNRPGC 99 TBMB 286 ± 109.76 98Sar₂-ACPWGPFWC(HyP)VNRPGC 100 TBMB 163.33 ± 38.41 99Sar₂-ACPWGPFWC(4FlPro)VNRPGC 101 TBMB 269.5 ± 6.86 100Sar₂-ACPWGPFWCP(tBuGly)NRPGC 102 TBMB 58.3 ± 50.37 112.45 ± 73.38 101Sar₂-ACPWGPFWCPVARPGC 103 TBMB 293 n = 1 265 ± 235.04 102Sar₂-ACPWGPFWCPV(D-Ala)RPGC TBMB 317 ± 168.56 311.67 ± 195.55 103Sar₂-ACPWGPFWCPVN(HArg)PGC 104 TBMB 126 ± 9.8 169.43 ± 94.28 104Sar₂-ACPWGPFWCPVNRAGC 105 TBMB 124 n = 1 193.67 ± 112.76 105Sar₂-ACPWGPFWCPVNR(D-Ala)GC TBMB 470.67 ± 221.53 106Sar₂-ACPWGPFWCPVNR(Aze)GC 106 TBMB 155 ± 47.04 107Sar₂-ACPWGPFWCPVNR(HyP)GC 107 TBMB 48.7 n = 1 85.83 ± 57.98 108Sar₂-ACPWGPFWCPVNR(Pip)GC 108 TBMB 374.5 ± 12.74 109Sar₂-ACPWGPFWCPVNR(4FlPro)GC 109 TBMB 184.5 ± 20.58 110Sar₂-ACPWGPFWCPVNR(Aib)GC 110 TBMB 75 ± 13.72 139.53 ± 103.98 111Sar₂-ACPWGPFWCPVNRPAC 111 TBMB 108 n = 1 237.5 ± 164.92 112Sar₂-ACPWGPFWCPVNRP(D-Ala)C TBMB 89 ± 15.68 113Sar₂-AC(Aib)(1Nal)GPFWCPVNRPGC 112 TBMB 10 n = 1 6.6 n = 1 114Sar₂-AC(Aib)WGPF(1Nal)CPVNRPGC 113 TBMB 21 n = 1 43 n = 1 115Sar₂-ACP(1Nal)GPFWCPV(D-Ala)RPGC TBMB 12.5 ± 0.98 1.64 ± 2.48 116Sar₂-ACP(1Nal)GPFWCPVNRP(D-Ala)C TBMB 2.95 ± 1.67 3.2 n = 1 117Sar₂-ACPWGPF(1Nal)CPV(D-Ala)RPGC TBMB 53 n = 1 75 n = 1 118Sar₂-ACPWGPF(1Nal)CPVNRP(D-Ala)C TBMB 37 n = 1 18 ± 13.72 119Sar₂-ACP(1Nal)G(Aib)FWCPVNRPGC 114 TBMB 21 n = 1 8.4 n = 1 120Sar₂-ACP(1Nal)GPF(1Nal)CPVNRPGC 115 TBMB 1.4 ± 0.39 0.98 n = 1 121Sar₂-ACP(1Nal)GPFWCP(tBuGly)NRPGC 116 TBMB 3.65 ± 0.29 2 n = 1 122Sar₂-ACP(1Nal)GPFWCPVN(HArg)PGC 117 TBMB 9.55 ± 0.69 8 n = 1 123Sar₂-ACPWG(Aib)F(1Nal)CPVNRPGC 118 TBMB 63 n = 1 46 n = 1 124Sar₂-AC(Aib)(1Nal)GPFWCPV(D-Ala)RPGC TBMB 26 n = 1 2.5 n = 1 125Sar₂-AC(Aib)(1Nal)GPFWCPVNRP(D-Ala)C TBMB 6.4 ± 0.78 0.61 ± 0.96 126Sar₂-ACP(1Nal)G(Aib)FWCP(tBuGly)NRPGC 119 TBMB 15 n = 1 19 n = 1 127Sar₂-ACP(1Nal)G(Aib)FWCPV(D-Ala)RPGC TBMB 40 n = 1 33 n = 1 128Sar₂-ACP(1Nal)G(Aib)FWCPVNRP(D-Ala)C TBMB 15 n = 1 16 n = 1 129Sar₂-ACP(1Nal)GPFWCP(tBuGly)(D-Ala)RPGC TBMB 23 n = 1 15 n = 1 130Sar₂-ACP(1Nal)GPFWCP(tBuGly)N(HArg)PGC 120 TBMB 0.29 ± 0.34 131Sar₂-ACP(1Nal)GPFWCP(tBuGly)NR(Aib)GC 121 TBMB 11 n = 1 6.8 n = 1 132Sar₂-ACP(1Nal)GPFWCP(tBuGly)NRP(D-Ala)C TBMB 7.7 ± 1.96 8.7 n = 1 133Sar₂-ACP(1Nal)GPFWCPV(D-Ala)(HArg)PGC TBMB 14 n = 1 3.7 n = 1 134Sar₂-ACP(1Nal)GPFWCPVN(HArg)P(D-Ala)C TBMB 1.2 n = 1 6.15 ± 0.29 135Sar₂-AC(Aib)(1Nal)G(Aib)FWCPVNR(Aib)GC 122 TBMB 43 n = 1 30 n = 1 136Sar₂-ACP(1Nal)G(Aib)FWCP(tBuGly)N(HArg)PGC 123 TBMB 23 n = 1 15 n = 1137 Sar₂-ACP(1Nal)G(Aib)FWCP(tBuGly)NR(Aib)GC 124 TBMB 20 n = 1 18 n = 1138 Sar₂-ACP(1Nal)GPFWCP(tBuGly)N(HArg)(Aib)GC 125 TBMB 5.1 n = 1

TABLE 7Biological Assay Data for Reference Peptide Ligands (Direct Binding Assay)Bicycle K_(D), nM ± 95% CI Compound Human Mouse Number Sequence ScaffoldEphA2 EphA2 139 AF488-(β-Ala)-Sar₁₀-H(D-Asp)VPCPWGPFWCPVNRPGCA TBMB0.31 ± 0.18 0.8 ± 0.54 140 AF488-(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB2.05 ± 0.62 4.55 ± 1.04C(Aib)(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)P(D-Asp)C 141AF488-(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 2.65 ± 0.64 6.5 ± 0.63C(Aib)(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR(Aib)(D-Asp)C 142Fl-(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.7 n = 1CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)P(D-Asp)C 143AF488-(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.4 ± 1.46 4.69 ± 4.15CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)P(D-Asp)C 144AF488-(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.04 n = 1 2.56 n = 1CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR(Aib)GC 145AF488-(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 2.17 ± 2.08 3.8 ± 0.55CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR(Aib)(D-Asp)C 146AF488-(β-Ala)-Sar₁₀-H(D-Asp)(C5g)P- TBMB 2.19 n = 1C(Aib)(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR(Aib)(D-Asp)C 147AF488-(β-Ala)-Sar₁₀-H(D-Asp)(C5g)P- TBMB 1.07 ± 0.9 3.44 ± 1.31CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)P(D-Asp)C

TABLE 8 Biological Assay Data for Reference Peptide Ligands (CompetitionBinding Assay) Ki, nM ± 95% CI Human Mouse EphA2 EphA FluorescentBicycle peptide Com- Com- Com- pound pound pound Number SequenceScaffold 18 18 148 Ac-Sar₂-ADVH- TBMB 1.2 ± 0.2 CPWGPFWCPVNRPGC (SEQ IDNO: 126) 149 ADVH-CP(3,3-DPA) TBMB 52.8 ± 11.6 GPFWCPVNRPGCA (SEQ ID NO:127) 150 ADVH-CP(1Nal) TBMB 0.12 ± 0.07 GPFWCPVNRPGCA (SEQ ID NO: 128)151 ADVH- TBMB 393.5 ± CPWAPFWCPVNRPGCA 206.78 (SEQ ID NO: 129) 152ADVH- TBMB  1.8 ± 0.74 CPWGAFWCPVNRPGCA (SEQ ID NO: 130) 153ADVH-CPWG(Aib) TBMB 0.51 ± 0.29 FWCPVNRPGCA (SEQ ID NO: 131) 154 ADVH-TBMB 101.03 ± CPWGPFWCAPVNRPGCA 33.68 (SEQ ID NO: 132) 155ADVH-CPWGPFWCPV TBMB 2 ± (D-Ala)RPGCA 0.74 156 ADVH-CPWGPFWCPVN TBMB14.93 ± (D-Ala)PGCA 2.3 157 Ac-Sar₂-ADVT- TBMB 0.91 ± 0.19CPWGPFWCPVNRPGC (SEQ ID NO: 133) 158 Ac-Sar₂-A(D-Asp)VT- TBMB 2.05 ±0.42  2.2 ± CPWGPFWCPVNRPGC 0.4 159 Ac-Sar₂-A(D-Asp)(D-Asp)T- TBMB 2.85± 0.49 CPWGPFWCPVNRPGC 160 Ac-Sar₂-A(D-Asp)(Cba)T- TBMB  2.6 ± 0.11CPWGPFWCPVNRPGC 161 Ac-Sar₂-A(D-Asp)(Cpa)T- TBMB 4.44 ± 1.08CPWGPFWCPVNRPGC 162 Ac-Sar₂-A(D-Asp)(Cpg)T- TBMB 2.55 ± 0.55CPWGPFWCPVNRPGC 163 Ac-Sar₂-A(D-Asp)(C5g)VT- TBMB 1.33 ± 0.27 1.74 ±CPWGPFWCPVNRPGC 1.23 164 Ac-Sar₂-AD(tBuGly)T- TBMB 2.25 ± 0.69CPWGPFWCPVNRPGC (SEQ ID: 134) 165 Ac-Sar₂-A(D-Asp)VT-C TBMB 185 ± 147(AC3C)WGPFWCPVNRPGC 166 Ac-Sar₂-A(D-Asp)VT-C TBMB 76.7 ±(AC4C)WGPFWCPVNRPGC 73.11 167 Ac-Sar₂-A(D-Asp)VT-C TBMB 138 n = 1(AC5C)WGPFWCPVNRPGC 168 Ac-Sar₂-A(D-Asp)VT-C TBMB 2 5.03 ± 2.24(4BenzyPro) WGPFWCPVNRPGC 169 Ac-Sar₂-A(D-Asp)VT-C TBMB 14.4 ± 7.64(4PhenyPro) WGPFWCPVNRPGC 170 Ac-Sar₂-A(D-Asp)VT-CP TBMB  0.6 ± 0.19(1Nal)GPFWCPVNRPGC 171 Ac-Sar₂-A(D-Asp)VT-CPWGP TBMB 4.88 ± 2.19(HArg)WCPVNRPGC 172 Ac-Sar₂-A(D-Asp)VT- TBMB 3.96 ± 0.72 CPWGPNWCPVNRPGC173 Ac-Sar₂-A(D-Asp)VT- TBMB 6.69 ± 3.49 CPWGPAWCPVNRPGC 174Ac-Sar₂-A(D-Asp)VT- TBMB  9.1 ± 1.73 CPWGPFWCPLNRPGC 175Ac-Sar₂-A(D-Asp)VT- TBMB 1.78 ± 0.54 CPWGPFWCPVN(HArg) P(D-Asp)C 176Ac-Sar₂-A(D-Asp)VT- TBMB 4.89 ± 0.97 CPWGPFWCPVN(HArg) P(D-Asp)C 177Ac-Sar₂-A(D-Asp)VT- TBMB 4.43 ± 2.37 CPWGPFWCPVNR (Aib)(D-Asp)C 178Ac-Sar₂-A(D-Asp)VT-CP(1Nal) TBMB  2.4 ± 0.23 G(Aib)FWCPVNR(Aib)GC 179Ac-Sar₂-A(D-Asp)VT-CPWG TBMB 2.94 ± 0.09 (Aib)F(1Nal)CPVNR(Aib)GC 180Ac-Sar₂-A(D-Asp)VT-CPWG TBMB 3.83 ± 0.43 (Aib)FWCP(tBuGly)NR (Aib)GC 181Ac-Sar₂-A(D-Asp)VT- TBMB 1.37 ± 0.41 CP(1Nal)G(Aib)F(1Nal) CPVNR(Aib)GC182 Ac-Sar₂-A(D-Asp)VT- TBMB 1.16 ± 0.39 CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)PGC 183 Ac-Sar₂-A(D-Asp)VT- TBMB 1.02 ± 0.41CP(1Nal)G(Aib)F(1Nal) CP(tBuGly)NR(Aib)GC 184 Ac-Sar₂-A(D-Asp)VT-CP TBMB1.19 ± 0.29 (1Nal)G(Aib)F(1Nal)CP (tBuGly)N(HArg)P(D-Asp)C 185(D-Asp)VT- TBMB 2.17 ± 0.73 CPWGPFWCPVNRPGC 186 (D-Asp)VT- TBMB 1.34 ±0.18 CP(1Nal)G(Aib)F(1Nal) CP(tBuGly)NR(Aib)GC 187 AHDVP-CP(1Nal) TBMB0.32 ± 0.03 GPFWCPVNRPGCA (SEQ ID NO: 135) 188 AHDVP-CP(1Nal) TBMB 1.45± 0.1  GPFWCPVNRPGC (SEQ ID NO: 136) 189 AHDVP-CPWGPF(1Nal) TBMB 1.3 ±0.2 CPVNRPGC (SEQ ID NO: 137) 190 AHDVP-CP(1Nal)GPFWCP TBMB 0.7 ± 0.4(tBuGly)NRPGC (SEQ ID NO: 138) 191 AHDVP-CP(1Nal)G(Aib) TBMB  3.1 ± 0.68FWCP(tBuGly)N(HArg)PGC (SEQ ID NO: 139) 192 AHDVP-CP(1Nal)G(Aib) TBMB1.75 ± 0.1  FWCP(tBuGly)NR(Aib)GC (SEQ ID NO: 140) 193 Ac-Sar₂-AHDVP-TBMB 0.59 ± 0.2  CPWGPFWCPVNRPGC (SEQ ID NO: 141) 194Ac-Sar₂-(D-Ala)HDVP- TBMB  1.2 ± 0.39 CPWGPFWCPVNRPGC 195 Ac-Sar₂-AADVP-TBMB 1.01 ± 0.19 CPWGPFWCPVNRPGC (SEQ ID NO: 142) 196Ac-Sar₂-A(D-His)DVP- TBMB 0.95 ± 0.24 CPWGPFWCPVNRPGC 197Sar₂-A(D-His)DVP- TBMB 1.2 CPWGPFWCPVNRPGC 198 Ac-Sar₂-A(D-His) TBMB 20± DVCPWGPFWCPVNRPGC 1.96 199 Sar₂-A(D-Ala)DVP- TBMB 3.35 ± 1.47CPWGPFWCPVNRPGC 200 Ac-Sar₂-A(D-Asp)DVP- TBMB 4.1 ± 0.2 CPWGPFWCPVNRPGC201 Sar₂-A(Thi)DVP- TBMB  0.6 ± 0.04 CPWGPFWCPVNRPGC (SEQ ID NO: 143)202 Sar₂-A(ThiAz)DVP- TBMB  0.7 ± 0.08 CPWGPFWCPVNRPGC (SEQ ID NO: 144)203 Sar₂-A(2FuAla)DVP- TBMB 0.49 ± 0.24 CPWGPFWCPVNRPGC (SEQ ID NO: 145)204 Ac-Sar₂-A(D-His)D(tBuGly) TBMB 2.15 ± 0.1  P-CPWGPFWCPVNRPGC 205Sar₂-AHAVP- TBMB 1.8 ± 0.2 CPWGPFWCPVNRPGC (SEQ ID NO: 146) 206Sar₂-AH(D-Ala)VP- TBMB  8.3 ± 0.78 CPWGPFWCPVNRPGC 207 Sar2-AHEVP- TBMB 1.3 ± 0.39 CPWGPFWCPVNRPGC (SEQ ID NO: 147) 208 Sar₂-AH(D-Glu)VP- TBMB2 ± CPWGPFWCPVNRPGC 0.39 209 Sar₂-AH(D-Asp)VP- TBMB 1.25 ± 0.29CPWGPFWCPVNRPGC 210 Ac-Sar₂-AH(D-Asp)VP- TBMB 1.1 ± 0.2 CPWGPFWCPVNRPGC211 Ac-Sar₂-AH(D-Asp)(tBuGly) TBMB 3.1 ± 0.2 P-CPWGPFWCPVNRPGC 212Ac-Sar₂-AH(D-Asp)V(Sar)- TBMB 4.95 ± 1.86 CPWGPFWCPVNRPGC 213Ac-Sar₂-AH(D-Asp)V(Aib) TBMB 1.9 ± 0.2 CPWGPFWCPVNRPGC 214 Sar₂-AHDAP-TBMB 22.5 ± 2.94 CPWGPFWCPVNRPGC (SEQ ID NO: 148) 215 Sar₂-AHD(D-Ala)P-TBMB 26 ± CPWGPFWCPVNRPGC 7.84 216 Sar₂-AHD(Aib)P- TBMB 2.77 ± 0.24CPWGPFWCPVNRPGC (SEQ ID NO: 149) 217 Sar₂-AHD(tBuGly)P- TBMB 0.49 n = 1CPWGPFWCPVNRPGC (SEQ ID NO: 150) 218 Sar₂-AHDVA- TBMB 1.27 ± 0.07CPWGPFWCPVNRPGC (SEQ ID NO: 151) 219 Sar₂-AHDV(D-Ala)- TBMB 15 ±CPWGPFWCPVNRPGC 3.92 220 Sar₂-AHDV(Aib)- TBMB 0.83 ± 0.15CPWGPFWCPVNRPGC (SEQ ID NO: 152) 221 Sar₂-AHDV(Aze)- TBMB  3.1 ± 0.39CPWGPFWCPVNRPGC (SEQ ID NO: 153) 222 Sar₂-AHDV(Pip)- TBMB 3.4 ± 0.2CPWGPFWCPVNRPGC (SEQ ID NO: 154) 223 (β-Ala)-Sar₁₀-HDVP- TBMB 1.29 ±0.42 CPWGPFWCPVNRPGC (SEQ ID NO: 155) 224 Ac-Sar₂-(D-His)DVP- TBMB 1.09± 0.13 CPWGPFWCPVNRPGC 225 Ac-Sar₂-H(D-Asp)VP- TBMB 1 ± 2.08 ±CPWGPFWCPVNRPGC 0.18 1.27 226 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 0.84 ± 0.24CPWGPFWCPVNRPGC 227 Ac-Sar₂-AH(D-Asp)VP- TBMB 0.75 ± 0.36 CP(1Nal)GPFWCP(tBuGly)N(HArg)PGC 228 Ac-Sar₂-A(D-His)DVP- TBMB 210.5 ±CPWGP(ΨAla)WCPVNRPGC 48.02 229 Ac-Sar₂-A(D-His)DVP- TBMB  5.1 ± 1.18CPWGPFWCP(HArg)NRPGC 230 Ac-Sar₂-A(D-His)DVP- TBMB  1.8 ± 0.78CP(1Nal)GPFWCP(tBuGly) N(HArg)PGC 231 Ac-Sar₂-A(D-His)DVP- TBMB 1.93 ±0.23 CP(1Nal)G(Aib)F(1Nal) CP(tBuGly)N(HArg)PGC 232 Ac-Sar₂-A(D-His)DVP-TBMB  0.9 ± 0.68 CP(1Nal)G(Aib)F(1Nal) CP(tBuGly)NR(Aib)GC 233Ac-Sar₂-A(D-His)DVP- TBMB  4.8 ± 0.84 CPWG(Aib)FWCP (tBuGly)NR(Aib)GC234 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 3.94 ± 1.72 C(Aib)(1Nal)G(Aib)F(1Nal)CP(tBuGly)NRPGC 235 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 2.58 ± 0.96C(Aib)(1Nal)G(Aib)F(1Nal) CP(tBuGly)N(HArg)PGC 236(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 3 ± C(Aib)(1Nal)G(Aib)F(1Nal) 0.71CP(tBuGly)N(HArg) P(D-Asp)C 237 Ac-(β-Ala)-Sar₁₀-H(D-Asp) TBMB 2.4 n = 1VP-C(Aib)(1Nal)G(Aib)F (1Nal)CP(tBuGly)N(HArg) P(D-Asp)C 238(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 2.83 ± 0.19 C(Aib)(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)(Aib) (D-Asp)C 239 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 4.91± 2.45 C(Aib)(1Nal)G(Aib)F(1Nal) CP(tBuGly)NR(Aib)GC 240(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 2.41 n = 1 C(Aib)(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR(Aib)(D-Asp)C 241 Ac-(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 2.98 ±0.96 C(Aib)(1Nal)G(Aib)F(1Nal) CP(tBuGly)NR(Aib)(D-Asp)C 242(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 7.77 ± 3.02 C(Aib)(1Nal)G(Aib)FWCP(tBuGly)N(HArg)P(D-Asp)C 243 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 2.83 ± 0.72C(Aib)(1Nal)GP(HArg)(1Nal) CP(tBuGly)NR(Aib)GC 244(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB  2.8 ± 0.26 C(Aib)(1Nal)GP(HArg)(1Nal)CP(tBuGly)N(HArg)P (D-Asp)C 245 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 11.91 ±C(Aib)WGP(HArg)(1Nal) 4.3 CP(tBuGly)N(HArg)P (D-Asp)C 246(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 31.4 ± 24.3 C(Aib)WGP(HArg)WCP(tBuGly)N(HArg)P(D-Asp)C 247 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 29.7 ±C(Aib)WGP(HArg)WCP 11.76 (tBuGly)N(HArg)P(D-Asp)C 248(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 28.4 ± 0.78 C(D-Ala)(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)P(D-Asp)C 249 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.38 ±0.46 CP(1Nal)G(Aib)(HArg)(1Nal) CP(tBuGly)NR(Aib)GC 250(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.48 ± 0.7  CP(1Nal)G(Aib)(HArg)(1Nal)CP(tBuGly)NR(Aib)(D-Asp)C 251 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.93 ± 0.62CP(1Nal)G(Aib)(HArg)(1Nal) CP(tBuGly)N(HArg) P(D-Asp)C 252(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 0.37 ± 0.18 CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)NRPGC 253 Ac-Sar₂-H(D-Asp)VP- TBMB 0.85 ± 0.82CP(1Nal)G(Aib)F(1Nal) CP(tBuGly)N(HArg)PGC 254 Ac-Sar₂-H(D-Asp)VP- TBMB0.74 ± 0.2  0.64 ± CP(1Nal)G(Aib)F(1Nal) 0.28 CP(tBuGly)N(HArg)P(D-Asp)C 255 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.53 ± 0.58CP(1Nal)G(Aib)F(1Nal)CP (tBuGly)N(HArg)P(D-Asp)C 256Ac-(β-Ala)-Sar₁₀-H(D-Asp) TBMB 0.41 n = 1 VP-CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)P(D-Asp)C 257 (β-Ala)-Sar₁₀-H(D-Asp) TBMB 1.07 ± 0.2 VP-CP(1Nal)G(Aib)F(1Nal) CP(tBuGly)N(HArg)PGC 258Ac-(β-Ala)-Sar₁₀-H(D-Asp) TBMB 0.54 n = 1 VP-CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR(Aib)GC 259 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 0.91 ± 0.14CP(1Nal)G(Aib)F(1Nal)CP (tBuGly)N(HArg)(Aib) (D-Asp)C 260Ac-(β-Ala)-Sar₁₀-H(D-Asp) TBMB 0.75 ± 0.07 VP-CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR (Aib)(D-Asp)C 261 Ac-Sar₂-H(D-Asp)VP- TBMB 0.63 ±0.43 CP(1Nal)G(Aib)F(1Nal) CPVNR(Aib)GC 262 Ac-Sar₂-H(D-Asp)VP- TBMB0.71 ± 0.17 0.72 ± CP(1Nal)G(Aib)F(1Nal) 0.31 CP(tBuGly)NR(Aib)GC 263(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 0.73 ± 0.26 CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR(Aib)GC 264 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 0.61 ± 0.31CP(1Nal)G(Aib)F(1Nal) CP(tBuGly)NR (Aib)(D-Asp)C 265(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.55 ± 0.34 CP(1Nal)G(Aib)FWCP(tBuGly)N(HArg) P(D-Asp)C 266 Ac-Sar₂-H(D-Asp)VP- TBMB  1.6 ± 0.63CP(1Nal)G(Aib) FWCPVNR(Aib)GC 267 Ac-Sar₂-H(D-Asp)VP-CPW TBMB 0.66 ±0.2  (Aza-Gly)PFWCPVNRPGC 268 Ac-Sar₂-H(D-Asp)VP-CPWG TBMB 1.24 ± 0.46(Aib)F(1Nal)CPVNR(Aib)GC 269 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 1.11 ± 0.08CPWG(Aib)F(1Nal)CP (tBuGly)N(Harg)P(D-Asp)C 270 Ac-Sar₂-H(D-Asp)VP- TBMB1.52 ± 1.27 CPWG(Aib)FWCP(tBuGly) NR(Aib)GC 271(β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 3.66 ± 1.7  CPWG(Aib)FWCP(tBuGly)N(HArg)P(D-Asp)C 272 (β-Ala)-Sar₁₀-H(D-Asp)VP- TBMB 3.99 ± 0.13CPWGP(HArg)WCP(tBuGly) N(HArg)P(D-Asp)C 273 (β-Ala)-Sar₁₀-H(D-Asp)(C5g)TBMB 1.5 n = 1 P-C(Aib)(1Nal)G(Aib)F(1Nal) CP(tBuGly)NR(Aib)(D-Asp)C 274Ac-(β-Ala)-Sar₁₀-H(D-Asp) TBMB 2.28 ± 0.69 (C5g)P-C(Aib)(1Nal)G(Aib)F(1Nal)CP (tBuGly)NR(Aib)(D-Asp)C 275 (β-Ala)-Sar₁₀-H(D-Asp)(C5g)TBMB 15.9 ± 0.2  P-C(Aib)WG(Aib)FWCP (tBuGly)NR(Aib)(D-Asp)C 276(β-Ala)-Sar₁₀-H(D-Asp)(C5g) TBMB 0.62 ± 0.27 P-CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)N(HArg)P(D-Asp)C 277 Ac-(β-Ala)-Sar₁₀-H(D-Asp) TBMB 0.53 ± 0.15(C5g)P-CP(1Nal)G(Aib)F (1Nal)CP(tBuGly)N (HArg)P(D-Asp)C 278(β-Ala)-Sar₁₀-H(D-Asp)(C5g) TBMB 0.46 ± 0.22 P-CP(1Nal)G(Aib)F(1Nal)CP(tBuGly)NR(Aib)GC 279 (β-Ala)-Sar₁₀-H(D-Asp)(C5g) TBMB 0.59 ± 0.28T-CP(1Nal)G(Aib)F(1Nal) CP(tBuGly)NR(Aib)GC 280 (β-Ala)-Sar₁₀-H(D-Asp)TBMB 0.64 ± 0.48 (C5g)T-CP(1Nal)G(Aib)F (1Nal)CP(tBuGly)N(HArg)P(D-Asp)C 281 Ac-Sar₂-A(D-Asp)DVT- TBMB 5.78 ± 1.1 CPWGPFWCPVNRPGC 282 Ac-Sar₂-A(D-Asp) TBMB 0.87 ± 0.14 DVT-CP(1Nal)GPFWCPVNRPGC 283 Ac-Sar₂-A(D-Asp)DVT-CP TBMB 0.28 ± 0.08(1Nal)GPF(1Nal)CPVNRPGC 284 Ac-Sar₂-A(D-Asp)DVT- TBMB  3.8 ± 0.77CP(1Nal)G(Aib)FWCP (tBuGly)N(HARrg)PGC 285 Ac-Sar₂-H(D-Asp)VT- TBMB 1.21± 0.29 CP(1Nal)G(Aib)F(1Nal)CP (tBuGly)NR(Aib)GC

TABLE 9 Biological Assay Data for Reference Peptide Ligands (Ala scanpeptides, Competition Binding Assay) Ki, nM ± 95% CI Human EphA2Fluorescent Bicycle peptide Com- Com- Com- pound pound pound NumberSequence Scaffold 66 18 25 Ac- TBMB 328 CANDWLCSLGWTC n = 1 (SEQ ID NO:27) 26 Ac- TBMB   71.6 ± CMNDWLCALGWTC 3.33 (SEQ ID NO: 28) 27 Ac- TBMB356 CMNDWLCSAGWTC n = 1 (SEQ ID NO: 29) 286 Sar₂- TBMB    886 ±ACAWGPFWCPVNRPGC 474.47 (SEQ ID NO: 156) 287 Sar₂- TBMB >11000ACPAGPFWCPVNRPGC (SEQ ID NO: 157) 288 Sar₂- TBMB >28000 >1000ACPWAPFWCPVNRPGC (SEQ ID NO: 158) 289 Sar₂- TBMB   1102 ± >1000ACPWGAFWCPVNRPGC 186.2 (SEQ ID NO: 159) 83 Sar₂- TBMB  287.5 ± 199ACPWGPAWCPVNRPGC 197.95 n = 1 (SEQ ID NO: 85) 290 Sar₂- TBMB >7000ACPWGPFACPVNRPGC (SEQ ID NO: 160) 291 Sar₂- TBMB >6000 >1000ACPWGPFWCAVNRPGC (SEQ ID NO: 161) 292 Sar₂- TBMB  953.5 ±ACPWGPFWCPANRPGC 59.78 (SEQ ID NO: 162) 101 Sar₂- TBMB    265 ± 293ACPWGPFWCPVARPGC 235.04 n = 1 (SEQ ID NO: 103) 293 Sar₂- TBMB    711 ±ACPWGPFWCPVNAPGC 581.64 (SEQ ID NO: 163) 104 Sar₂- TBMB 193.67 ± 124ACPWGPFWCPVNRAGC 112.76 n = 1 (SEQ ID NO: 105) 111 Sar₂- TBMB  237.5 ±108 ACPWGPFWCPVNRPAC 164.92 n = 1 (SEQ ID NO: 111) 294 Sar₂-AC(D-Ala)TBMB >4000 WGPFWCPVNRPGC 295 Sar₂-ACP(D-Ala) TBMB >7000 GPFWCPVNRPGC 296Sar₂-ACPW(D-Ala) TBMB 1003 PFWCPVNRPGC n = 1 297 Sar₂-ACPWG(D-Ala) TBMB1497 FWCPVNRPGC n = 1 298 Sar₂-ACPWGP(D-Ala) TBMB >6500 WCPVNRPGC 299Sar₂-ACPWGPF(D-Ala) TBMB >4000 CPVNRPGC 300 Sar₂-ACPWGPFC(D-Ala)TBMB >1200 VNRPGC 301 Sar₂-ACPWGPFWCP TBMB >4000 (D-Ala)NRPGC 102Sar₂-ACPWGPFWCPV TBMB 311.67 ±    317 ± (D-Ala)RPGC 195.55 168.56 302Sar₂-ACPWGPFWCPVN TBMB   1410 ± >1000 (D-Ala)PGC 680.11 105Sar₂-ACPWGPFWCPVNR TBMB 470.67 ±  677 (D-Ala)GC 221.53 n = 1 112Sar₂-ACPWGPFWCPVNRP TBMB 109.83 ±     89 ± (D-Ala)C 66.19 15.68 303(β-Ala)-Sar₁₀- TBMB >1000 ACWAPFWCAVNRPGC (SEQ ID NO: 164) 3044-(pyridyl-2-disulfanyl)- TBMB >10000 4-RS-methylbutanoyl-(β-Ala)-Sar₁₀- ACPWAPFWCAVNRPGC (SEQ ID NO: 165) 173 Ac-Sar₂-A(D-Asp)TBMB   6.69 ± VTCPWGPAWCPVNRPGC 3.49 305 (β-Ala)-Sar₁₀-H(D-Asp)TBMB >5000 VPCP(1Nal)A(Aib)F(1Nal) CA(tBuGly)NR(Aib) (D-Asp)C 151ADVHCPW(Ala) TBMB  393.5 ± PFWCPVNRPGCA 206.78 (SEQ ID NO: 166) 152ADVHCPWG(Ala) TBMB 1.8 ± FWCPVNRPGCA 0.74 (SEQ ID NO: 167) 154ADVHCPWGPFWC TBMB 101.03 ± (D-Ala)VNRPGCA 33.68 155 ADVHCPWGPFWCPV TBMB2 ± (D-Ala)RPGCA 0.74 156 ADVHCPWGPFWCPVN TBMB  14.93 ± (D-Ala)PGCA 2.3306 DOTA-(β-Ala)-Sar₁₀-H TBMB >250 (D-Asp)VPCP(1Nal)A(Aib)F(1Nal)CA(tBuGly) NR(Aib)(D-Asp)C

TABLE 10 Biological Assay Data for Reference Peptide Ligands (Ala scanpeptides, Direct Binding Assay) K_(D), nM ± Bicycle 95% CI CompoundHuman Number Sequence Scaffold EphA2 307 AF488-(β-Ala)-Sar₁₀- TBMB >1000ACPWAPFWCAVNRPGC (SEQ ID NO: 168) 308 AF488-(β-Ala)-Sar₁₀-H(D-Asp)TBMB >2000 VPCP(1Nal)A(Aib)F(1Nal) CA(tBuGly)NR(Aib)(D-Asp)C

Reference Examples B1-B98

The following reference peptide ligands having a TATA scaffold withthree thioether linkages to cysteine residues of the specified peptidesequences were prepared and evaluated for affinity to EphA2 as describedin detail in our earlier application GB201721259.8 filed 19 Dec. 2017.

In view of the results obtained above in Examples 1-6, it is predictedthat derivatives of the reference examples B1-B98 according to thepresent invention, i.e. having alkylamino linkages in place of one ormore of the thioether linkages in the reference examples, will alsodisplay affinity for EphA2. It is further predicted that derivatives ofthe reference examples B1-B98 having scaffolds other than TATA, inparticular non-aromatic scaffolds other than TATA, will also displayaffinity for EphA2. All such derivatives having affinity for EphA2 aretherefore included within the scope of the present invention.

TABLE 11 Biological Assay Data for Reference Peptide Ligands (TATApeptides, Direct Binding Assay) Bicycle Com- SEQ Human pound ID EphA2(K_(D), Number Sequence NO: Scaffold nM ± 95% CI) 1 ACMNDWWCAMGWKCA- 169TATA   304 ± 91.99 Sar₆-K(Fl) 2 ACVPDRRCAYMNVCA- 170 TATA 74.91 ± 6.6  Sar₆-K(Fl) 3 ACVVDGRCAYMNVCA- 171 TATA 129.8 ± 80.75 Sar₆-K(Fl) 4ACVVDSRCAYMNVCA- 172 TATA 124.6 ± 51.74 Sar₆-K(Fl) 5 ACVPDSRCAYMNVCA-173 TATA 93.95 ± 23.62 Sar₆-K(Fl) 6 ACYVGKECAIRNVCA- 174 TATA 168.5 ±20.58 Sar₆-K(Fl) 7 ACYVGKECAYMNVCA- 175 TATA 149.73 ± Sar₆-K(Fl) 39.2 8Fl-G-Sar₅- 176 TATA 218.33 ± ACYVGKECAYMNVCA 10.51 9 Fl-(β-Ala)-Sar₁₀-177 TATA 6.43 ± 1.15 ARDCPLVNPLCLHPGWTC 10 Fl-(β-Ala)-Sar₁₀-A(HArg) 178TATA 9.07 ± 2.49 DCPLVNPLCLHPGWTC 11 Ac- TATA 3.08 ± 0.43CPLVNPLCLHPGWTCLHG- Sar₆-(D-K[Fl]) 12 Ac-CPLVNPLCLHPGWTCL TATA 10.56 ±0.77  (D-His)G-Sar₆-(D-K[Fl]) 13 Ac- TATA 5.29 ± 0.79CPLVNPLCLHPGWSCRGQ- Sar₆-(D-K[Fl]) 14 Ac-CPLVNPLCLHPGWSC TATA 9.96 ±0.55 (HArg)GQ-Sar₆-(D-K[Fl])

TABLE 12 Biological Assay Data for Peptide Ligands of the Invention(TATA peptides, Competition Binding Assay) Ki, nM ± 95% CI FluorescentPeptide Human EphA2 Mouse EphA2 Bicycle SEQ Reference ReferenceReference Reference Compound ID Compound Compound Compound CompoundNumber Sequence NO: Scaffold C B A C 15 ACMNDWWCAMGWKCA 179 TATA 277.5 ±38.22 16 ACVPDRRCAYMNVCA 180 TATA 69.97 ± 8.67  17(β-Ala)-Sar₁₀-ACVPDRRCAYMNVC 181 TATA 85.05 ± 1.08  18 DLRCGGDPRCAYMNVCA182 TATA 70.8 ± 2.35 19 SRPCVIDSRCAYMNVCA 183 TATA 94.75 ± 24.01 20ESRCSPDARCAYMNVCA 184 TATA 57.05 ± 4.61  21 HSGCRPDPRCAYMNVCA 185 TATA62.15 ± 4.61  22 GSGCKFDSRCAYMNVCA 186 TATA 63.25 ± 13.82 23ETVCLPDSRCAYMNVCA 187 TATA   130 ± 15.68 24 GQVCIVDARCAYMNVCA 188 TATA168.5 ± 16.66 25 ACVPDRRCAFENVCVDH 189 TATA 97.3 ± 3.33 26ACVPDERCAFMNVCEDR 190 TATA 39.05 ± 10.29 27 ACVPDRRCAFQDVCDHE 191 TATA 159 n = 1 28 ACVPDRRCAFRDVCLTG 192 TATA 1700 n = 1  29 ACYVGKECAYMNVCA193 TATA  209.5 ± 110.74 106.65 ± 24.94 87.7 n = 1 30 ACQPSNHCAFMNYCA194 TATA  293 n = 1 186.53 ± 86.86  137 n = 1 31 ACSPTPACAVQNLCA 195TATA  223 n = 1   177 ± 60.76 32 ACTSCWAYPDSFCA 196 TATA   232 ± 52.19 151 n = 1 33 ACTKPTGFCAYPDTICA 197 TATA 268.5 ± 16.66 34ACRGEWGYCAYPDTICA 198 TATA 347.5 ± 57.82 35 ACRNWGMYCAYPDTICA 199 TATA282.5 ± 65.66 36 ACPDWGKYCAYPDTICA 200 TATA  160 ± 1.96 37ACRVYGPYCAYPDTICA 201 TATA 294.5 ± 20.58 38 ACSSCWAYPDSVCA 202 TATA400.33 ± 205.19 39 ACQSCWAYPDTYCA 203 TATA 321.33 ± 119.53 40ACGFMGLEPCETFCA 204 TATA 187.5 ± 20.58 41 ACGFMGLVPCEVHCA 205 TATA 155 ±9.8  42 ACGFMGLEPCEMVCA 206 TATA 320.5 ± 14.7  43 ACGFMGLEPCVTYCA 207TATA 233.5 ± 20.58 44 ACGFMGLEPCELVCA 208 TATA 126.8 ± 21.17 45ACGFMGLVPCNVFCA 209 TATA   142 ± 41.16 46 ACGFMGLEPCELFCA 210 TATA 81.7± 7.06 47 ACGFMGLEPCELFCMPK 211 TATA   185 ± 74.48 48 ACGFMGLEPCELYCA212 TATA 127.5 ± 14.7  49 ACGFMGLEPCELYCAHT 213 TATA   144 ± 17.64 50ACGFMGLEPCEMYCA 214 TATA   140 ± 45.08 51 ACGFMGLVPCELYCADN 215 TATA 84.4 ± 36.46 52 ACPLVNPLCLTSGWKCA 216 TATA 115.33 ± 11.33  53ACPMVNPLCLHPGWICA 217 TATA 15.4 ± 3.17 54 ACPLVNPLCLHPGWICA 218 TATA15.25 ± 2.84  55 ACPLVNPLCLHPGWRCA 219 TATA 20.55 ± 0.88  56ACPLVNPLCNLPGWTCA 220 TATA   184 ± 115.64 57 ACPLVNPLCLVPGWSCA 221 TATA35.4 ± 10   58 ACPLVNPLCLLDGWTCA 222 TATA 38.35 ± 5.39  59ACPLVNPLCLMPGWGCA 223 TATA 114.5 ± 10.78 60 ACPLVNPLCMIGNWTCA 224 TATA96.2 ± 0.59 61 ACPLVNPLCLMTGWSCA 225 TATA 241.5 ± 44.1  62ACPLVNPLCMMGGWKCA 226 TATA  67.1 ± 19.21 63 ACPLVNPLCLYGSWKCA 227 TATA59.05 ± 28.32 64 ACPLVNPLCLHPGWTCA 228 TATA   30 n = 1 65ARDCPLVNPLCLHPGWTCA 229 TATA 6.05 ± 1.38 39.1 ± 0.39 66 (β-Ala)-Sar₁₀-230 TATA 4.94 ± 1.41  57.6 ± 24.86 (BCY6099) ARDCPLVNPLCLHPGWTC 67(β-Ala)-Sar₁₀- 231 TATA 8.51 ± 0.17  61.7 ± 15.48 (BCY6014)A(HArg)DCPLVNPLCLHPGWTC 68 Ac-ARDCPLVNPLCLHPGWTCA- TATA 19.3 ± 4.92166.5 ± 30.38 Sar₆-(D-K) 69 Ac- TATA 17.5 ± 0.98 164.5 ± 2.94 A(HArg)DCPLVNPLCLHPGWTCA- Sar₆-(D-K) 70 RPACPLVNPLCLHPGWTCA 232 TATA10.06 ± 2.96  71 RPPCPLVNPLCLHPGWTCA 233 TATA 11.11 ± 2.25  72KHSCPLVNPLCLHPGWTCA 234 TATA 11.92 ± 6.04  73 ACPLVNPLCLHPGWTCLHG 235TATA 1.98 ± 0.49 7.27 ± 1.09 74 Ac-CPLVNPLCLHPGWTCLHG 236 TATA 1.76 ±0.54 75 (β-Ala)-Sar₁₀- 237 TATA 2.48 ± 0.27   18 ± 1.18ACPLVNPLCLHPGWTCLHG 76 (β-Ala)-Sar₁₀- TATA 10.01 ± 1.55  75.15 ± 14.41ACPLVNPLCLHPGWTCL(D-His)G 77 Ac-CPLVNPLCLHPGWTCLHG- TATA 5.41 ± 0.8648.23 ± 15.72 (BCY6019) Sar₆-(D-K) 78 Ac-CPLVNPLCLHPGWTCL(D- TATA 15.6 ±4.7  115.03 ± 41.16  His)G-Sar₆-(D-K) 79 ACPLVNPLCLHPG(2Nal)TCLHG 238TATA   162 ± 17.64 80 RHDCPLVNPLCLLPGWTCA 239 TATA 7.11 ± 0.72 81TPRCPLVNPLCLMPGWTCA 240 TATA  9.8 ± 2.61 82 ACPLVNPLCLAPGWTCA 241 TATA46.2 n = 1 83 ACPLVNPLCLAPGWTCSRS 242 TATA 7.05 ± 1.11 84ACPLVNPLCLEPGWTCA 243 TATA 53.9 n = 1 85 ACPLVNPLCLEPGWTCAKR 244 TATA10.95 ± 1.6   86 ACPLVNPLCLHPGWSCA 245 TATA 56.15 ± 11.27 87ACPLVNPLCLHPGWSCRGQ 246 TATA 2.57 ± 0.63 18.6 ± 0.59 (BCY6026) 88Ac-CPLVNPLCLHPGWSCRGQ 247 TATA 1.64 ± 0.75 89 (β-Ala)-Sar₁₀- 248 TATA2.86 ± 1.29 29.55 ± 4.61  ACPLVNPLCLHPGWSCRGQ 90 (β-Ala)-Sar₁₀- 249 TATA5.41 ± 0.67 47.05 ± 11.47 ACPLVNPLCLHPGWSC(HArg)GQ 91Ac-CPLVNPLCLHPGWSCRGQ- TATA 5.98 ± 1.42 49.87 ± 14.44 (BCY6042)Sar₆-(D-K) 92 Ac- TATA 10.56 ± 6.56  75.27 ± 21.72CPLVNPLCLHPGWSC(HArg)GQ- Sar₆-(D-K) 93 ACPLVNPLCLHPG(2Nal)SCRGQ 250 TATA   228 ± 103.88 94 ACPLVNPLCLTPGWTCTNT 251 TATA 13.25 ± 4.05  95ACPMVNPLCLHPGWKCA 252 TATA 11.91 ± 3.73  96 ACPMVNPLCLTPGWICA 253 TATA16.07 ± 4.58  97 ACPMVNPLCLHPGWTCA 254 TATA   20 ± 1.02

TABLE 13 Biological Assay Data for Peptide Ligands of the Invention(Competition Binding Assay) Bicycle Human EphA2 Compound ReferenceNumber Sequence Scaffold Compound C 98 (β-Ala)-Sar₁₀-H(D-Asp)VT- TATA251.5 ± 73.5 C(Aib)(1Nal)G(Aib)F(1Nal)CP (tBuGly)N(HArg)P(D-Asp)C

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed aspects and embodiments of the present invention will beapparent to those skilled in the art without departing from the scope ofthe present invention. Although the present invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are apparent tothose skilled in the art are intended to be within the scope of thefollowing claims.

The invention claimed is:
 1. A peptide ligand specific for EphA2comprising a polypeptide comprising a sequence:[β-Ala][Sar]₁₀H[dD]VPA₁PWGPFWA₂PVNRPGA₃; or[β-Ala][Sar]₁₀H[dD]VPA₁[Aib][1Nal]G[Aib]F[1Nal]A₂P[tBuGly]N[HArg]P[dD]A₃;wherein A₁, A₂, and A₃ each represent independently a residue selectedfrom the group consisting of cysteine, L-2,3-diaminopropionic acid(Dap), N-beta-alkyl-L-2,3-diaminopropionic acid (N-AlkDap), andN-beta-haloalkyl-L-2,3-diaminopropionic acid (N-HAlkDap), with theproviso that at least one of the A₁, A₂, and A₃ is selected from thegroup consisting of Dap, N-AlkDap, and N-HAlkDap, the A₁, A₂, and A₃residues being separated by at least two loop sequences, and a molecularscaffold, the peptide being linked to the scaffold by covalentalkylamino linkages with the Dap or N-AlkDap or N-HAlkDap residues ofthe polypeptide and by thioether linkages with the cysteine residues ofthe polypeptide when the said three residues include cysteine, such thattwo polypeptide loops are formed on the molecular scaffold; wherein Aibrepresents aminoisobutyric acid, 1Nal represents 1-naphthylalanine,tBuGly represents tert-leucine, Sar represents sarcosine, HArgrepresents homoarginine, and dD represents D-aspartic acid.
 2. Thepeptide ligand as defined in claim 1, wherein two of A₁, A₂ and A₃ areselected from the group consisting of Dap, N-AlkDap, and N-HAlkDap, andthe third one of A₁, A₂ and A₃ is cysteine.
 3. The peptide ligand asdefined in claim 1, wherein A₁, A₂ and A₃ are each N-AlkDap orN-HAlkDap.
 4. The peptide ligand as defined in claim 1, wherein themolecular scaffold is an aromatic molecular scaffold.
 5. The peptideligand as defined in claim 4, wherein the peptide ligand comprises anamino acid sequence selected from the group consisting of:AF488-(β-Ala)-Sar₁₀-H(D-Asp)VPCPWGPFWCPVNRPGCA; and(β-Ala)-Sar₁₀-H(D-Asp)VP-CPWGPFWCPVNRPGC, or a pharmaceuticallyacceptable salt thereof, with the proviso that one or more of thecysteine residues in said amino acid sequence is replaced by Dap,N-AlkDap or N-HAlkDap.
 6. The peptide ligand as defined in claim 4,wherein the aromatic molecular scaffold is 1,3,5-tris(methylene)benzene.7. The peptide ligand as defined in claim 1, wherein A₂ is cysteine andA₁ and A₃ are each independently Dap, N-AlkDap or N-HAlkDap.
 8. Thepeptide ligand as defined in claim 7, wherein A₁ and A₃ are Dap.
 9. Thepeptide ligand as defined in claim 1, wherein the molecular scaffold isa non-aromatic molecular scaffold.
 10. The peptide ligand as defined inclaim 9, wherein the non-aromatic molecular scaffold is1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).
 11. Thepeptide ligand as defined in claim 1, wherein the EphA2 is human EphA2.12. The peptide ligand as defined in claim 1, wherein the peptide ligandis selective for human EphA2, but does not cross-react with human EphA1,EphA3 or EphA4.
 13. The peptide ligand as defined in claim 1, whereinone of A₁, A₂ and A₃ is selected from the group consisting of Dap,N-AlkDap, and N-HAlkDap.
 14. The peptide ligand as defined in claim 1,wherein the peptide ligand comprises an amino acid sequence selectedfrom the group consisting of:[β-Ala][Sar]₁₀H[dD]VP[Dap]PWGPFW[Dap]PVNRPGC;[β-Ala][Sar]₁₀H[dD]VPCPWGPFW[Dap]PVNRPG[Dap];[β-Ala][Sar]₁₀H[dD]VP[Dap]PWGPFWCPVNRPG[Dap];[β-Ala][Sar]₁₀H[dD]VP[Dap][Aib][1Nal]G[Aib]F[1Nal][Dap]P[tBuGly]N[HArg]P[dD]C;[β-Ala][Sar]₁₀H[dD]VPC[Aib][1Nal]G[Aib]F[1Nal][Dap]P[tBuGly]N[HArg]P[dD][Dap]; and [β-Ala][Sar]₁₀H[dD]VP[Dap][Aib][1Nal]G[Aib]F [1Nal]CP[tBuGly]N[HArg]P [dD][Dap], or apharmaceutically acceptable salt thereof.
 15. A drug conjugatecomprising a peptide ligand as defined in claim 1, conjugated to one ormore effector and/or functional groups.
 16. The drug conjugate asdefined in claim 15, wherein said effector and/or functional groupscomprise a cytotoxic agent selected from the group consisting of DM1 andMMAE.
 17. The drug conjugate as defined in claim 16, wherein thecytotoxic agent is MMAE.
 18. A pharmaceutical composition whichcomprises the drug conjugate of claim 15, in combination with one ormore pharmaceutically acceptable excipients.
 19. A pharmaceuticalcomposition which comprises the peptide ligand of claim 1, incombination with one or more pharmaceutically acceptable excipients.